Methods of diagnosing bladder cancer

ABSTRACT

The present invention relates to methods of diagnosing bladder cancer in a patient, involving determining the methylation status of Methylation Variable Positions (MVPs) in DNA from the patient and providing a diagnosis based on methylation status data. The invention also relates to methods of treating bladder cancer comprising providing a diagnosis of bladder cancer by the diagnostic methods defined herein followed by administering one or more anti-cancer agents to a patient. The invention also relates to methylation-discriminatory arrays comprising probes directed to the MVPs defined herein and kits comprising the arrays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371 that claims priority to International Application No. PCT/GB2016/051903 filed Jun. 24, 2016, which claims priority to Great Britain Patent Application No. 1511152.9 filed Jun. 24, 2015, all of which are incorporated herein reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of diagnosing bladder cancer in a patient, involving determining the methylation status of Methylation Variable Positions (MVPs) in DNA from the patient and providing a diagnosis based on methylation status data. The invention also relates to methods of treating bladder cancer comprising providing a diagnosis of bladder cancer by the diagnostic methods defined herein followed by administering one or more anti-cancer agents to a patient. The invention also relates to methylation-discriminatory arrays comprising probes directed to the MVPs defined herein and kits comprising the arrays.

BACKGROUND TO THE INVENTION

Bladder cancer represents one of the most common malignancies in the western world, ranking as the 5th most common cancer in the United States and causing approximately 3% of all cancer-related deaths [1, 2]. The foremost clinical sign at presentation is hematuria, and bladder cancer is detected in about 10% of all such cases investigated [3]. Bladder cancer is more likely in older male patients, current or past smokers and patients exposed to industrial carcinogens [4]. Younger females with nonvisible hematuria are less likely to harbor bladder cancer and for these patients delay in detection of bladder cancer, following misdiagnosis of haematuria, is a frequent event [5]. Cystoscopy is the current gold standard for detecting bladder cancer and is an invasive, uncomfortable procedure requiring clinic or hospital attendance and posing a small but significant risk of infection [6-9].

Each year in the UK, approximately 10,300 people are diagnosed with bladder cancer and 5,000 die from the disease. However, more than 100,000 cases per year are referred from primary care to urology haematuria clinics for cystoscopy and imaging. Bladder cancer is detected in only 10% of patients referred.

Of those with confirmed disease, two thirds are non-muscle invasive bladder cancer (NMIBC) and of these, 70% will recur and 15% will progress to muscle invasive bladder cancer (MIBC). Surveillance by cystoscopy is necessary to detect recurrence and is performed as frequently as 3 monthly for 2 years then 6 monthly and annually thereafter for cases at high risk of recurrence. The investigation of haematuria and the subsequent surveillance for recurrence poses a significant health economic cost estimated as £55.39 million, ranking bladder as one of the most expensive cancers to manage [10, 11]. There is therefore a great need for improved assays which can better identify patients harbouring disease and reduce the need for unnecessary cystoscopy.

No urinary-based biomarker has FDA approval as a standalone test for the detection of bladder cancer, and consequently guidelines recommend cystoscopy of all patients with visible haematuria and persistent nonvisible haematuria [10, 11]. Urine cytology is frequently used as a diagnostic aid in conjunction with cystoscopy but has low sensitivity to detect cancer other than high grade disease and carcinoma in situ, and cannot replace cystoscopy [12, 13]. Similarly commercially available assays based on single targets or small panels of targets fail to detect bladder cancer with sufficient sensitivity and are approved for use only in conjunction with cystoscopy [14].

Several studies have now shown the potential utility of DNA methylation biomarkers in body fluids, including urine [15-23], plasma/serum [24-26], and sputum [27, 28], for the non-invasive detection of cancer. Changes in DNA methylation play a key role in malignant transformation, leading to the silencing of tumor-suppressor genes and overexpression of oncogenes [29]. The ontogenic plasticity and relative stability of DNA methylation makes epigenetic changes ideal biomarkers for diagnosis.

Detection assays involving the presence of specific proteins in voided urine have been developed and commercialised. In these cases the number of proteins detected per assay is low and specificities and sensitivities remain unsatisfactory [14]. Detected protein biomarkers include human complement factor H-related protein, carcinoembryonic antigen (CEA), bladder tumor cell-associated mucins and nuclear mitotic apparatus protein 22 (NMP22).

In terms of assays assessing the expression of certain proteins, WO2014042763 describes a nine-biomarker panel consisting of IL-8, MMP9, SDC1, CCL18, SERPINE1, CD44, VEGF-A, CA9, and ANG for detection of protein in urine samples; a further nine-biomarker panel consisting of CA9, CCL18, MMP12, TMEM45A, MMP9, SEMA3D, ERBB2, CRH, and MXRA8; as well as a three-biomarker panel consisting of CCL18, CD44, and VEGF-A.

To date, DNA methylation biomarker assays for the detection of bladder cancer have been centered on the analysis of only a small number of loci, in part due to technological limitations and derivation of targets with cancer specificity [11-19]. In general, reported sensitivities and specificities are high relative to established assays, but would fail to attain performance characteristics achieved by cystoscopy. Methylation markers for bladder cancer previously studied include DAPK, BCL2, TERT, TWIST1, NID2, RARbeta, E-cadherin and p16. International patent application publication WO2013/144362 describes a diagnostic assay for bladder cancer involving detecting methylation of the promoter of the ECRG4 and/or the ITIH5 gene. US patent application publication US2013224738 describes a diagnostic assay for bladder cancer involving assessing the methylation status of genes consisting of BCL2, CDKN2A and NID2.

Improved assays for the accurate diagnosis of bladder cancer are sought and would be of significant clinical and economic benefit, particularly assays which are non-invasive.

SUMMARY OF THE INVENTION

Diagnostic methods are provided which can detect bladder cancer from a biological sample, particularly a voided urine sample, with robust and high sensitivity and specificity, and which have the potential to reduce the need for cystoscopy in patients referred with haematuria and in patients undergoing surveillance for disease recurrence. Avoiding cystoscopy will reduce the cost of bladder cancer management and positively impact on patient wellbeing, reducing both the number of hospital visits and the inconvenience of an invasive investigation. Thus the invention provides the following:

The invention provides a method of diagnosing bladder cancer in an individual comprising:

-   -   (a) providing DNA from a sample from the individual;     -   (b) determining whether each one of a group of MVPs selected         from a panel comprising the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG] is methylated, wherein the group         comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG]; and     -   (c) diagnosing bladder cancer in the individual when at least 25         of the MVPs of the group of (b) are methylated.

In any such method the group of MVPs may comprises at least 40 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], and wherein bladder cancer is diagnosed when at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated.

The group of MVPs may comprise at least 50 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], or may comprise at least 100 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

The group of MVPs may comprise all 150 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

In methods described above, cancer may be diagnosed in the individual when at least 40 of the MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated, or when at least 50 of the MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated, or when at least 100 of the MVPs are methylated, or when all 150 MVPs are methylated.

In methods described above, the MVPs determined to be methylated may include the MVPs identified in SEQ ID NOS 1 to 3 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 5 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 10 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 40 and denoted by [CG].

In methods described above, the group of MVPs may comprise all 150 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein bladder cancer is diagnosed in the individual when at least 40 of the MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated, and wherein the MVPs determined to be methylated include the MVPs identified in SEQ ID NOS 1 to 10 and denoted by [CG].

In any of the methods described above, the step of determining whether each one the MVPs is methylated may comprise bisulphite converting the DNA.

In any of the methods described above, the step of determining whether each one the MVPs is methylated may comprise:

-   -   1) performing a sequencing step to determine the sequence of         MVPs;     -   2) hybridising DNA to an array comprising probes capable of         discriminating between methylated and non-methylated forms of         MVPs and applying a detection system to the array to         discriminate methylated and non-methylated forms of the MVPs; or     -   3) performing an amplification step using methylation-specific         primers, wherein the status of an MVP as methylated or         non-methylated is determined by the presence or absence of an         amplified product.

Before the sequencing or hybridization steps, an amplification step may be performed, wherein loci comprising each MVP are amplified. Amplification may be performed by PCR.

A capturing step may be performed before the sequencing or hybridization steps. The capturing step may involve binding polynucleotides comprising the MVP loci to binding molecules specific to the MVP loci and collecting complexes comprising MVP loci and binding molecules; and wherein:

-   -   i. the capturing step occurs before the step of bisulphite         converting the DNA;     -   ii. the capturing step occurs after the step of bisulphite         converting the DNA but before the amplification or hybridization         steps; or     -   iii. the capturing step occurs after the step of bisulphite         converting the DNA and after the amplification step.

The binding molecules may be oligonucleotides specific for each MVP, preferably DNA or RNA molecules each comprising a sequence which is complementary to the corresponding MVP.

The binding molecule may be coupled to a purification moiety.

The purification moiety may comprise a first purification moiety and the step of collecting complexes comprising MVP loci and binding molecules may comprise binding the first purification moiety to substrates comprising a second purification moiety, wherein first and second purification moieties form an interaction complex.

The first purification moiety may be biotin and the second purification moiety may be streptavidin; or the first purification moiety may be streptavidin and the second purification moiety may be biotin.

The step of amplifying loci comprising MVPs may comprise the use of primers which are independent of the methylation status of the MVP.

The step of amplifying loci comprising MVPs may be performed by microdroplet PCR amplification.

In any of the methods described above, the biological sample obtained from the individual may be a sample of urine, blood, serum, plasma or cell-free DNA.

In any of the methods described above, the method may achieve a ROC sensitivity of 95% or greater and a ROC specificity of 90% or greater; preferably a ROC sensitivity of 96% and a ROC specificity of 97%, preferably a ROC AUC of 95% or greater, preferably 98%.

In any of the methods described above, the method may achieve a negative predictive value (NPV) of 95% or greater, preferably 97%.

In any of the methods described above, the step of diagnosing bladder cancer in the individual may further comprise:

-   -   I. stratifying the grade of the tumor; and/or     -   II. determining the risk of recurrence of the tumor; and/or     -   III. determining the risk of progression of non-muscular         invasive disease; and/or         determining the likely response to treatment therapy.

The invention additionally provides a method of treating bladder cancer in an individual comprising:

-   -   (a) obtaining DNA from a sample from the individual and         determining whether each one of a group of MVPs selected from a         panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG] is methylated, wherein the group comprises at         least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG];     -   (b) diagnosing bladder cancer in the individual when at least 25         MVPs of the group of (a) are methylated; and     -   (c) administering one or more bladder cancer treatments to the         individual.

The invention additionally provides a method of treating bladder cancer in an individual comprising:

-   -   (a) providing DNA from a sample from the individual and         determining whether each one of a group of MVPs selected from a         panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG] is methylated, wherein the group comprises at         least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG];     -   (b) diagnosing bladder cancer in the individual when at least 25         MVPs of the group of (a) are methylated; and     -   (c) administering one or more bladder cancer treatments to the         individual.

The invention additionally provides a method of treating bladder cancer in an individual comprising:

-   -   (a) determining whether each one of a group of MVPs selected         from a panel comprising the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG] is methylated in DNA from a sample from         the individual, wherein the group comprises at least 25 of the         MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG];     -   (b) diagnosing bladder cancer in the individual when at least 25         MVPs of the group of (a) are methylated; and     -   (c) administering one or more bladder cancer treatments to the         individual.

The invention additionally provides a method of treating bladder cancer in an individual comprising administering one or more bladder cancer treatments to the individual, wherein the individual has been diagnosed with bladder cancer by steps comprising:

-   -   (a) providing DNA from a sample from the individual and         determining whether each one of a group of MVPs selected from a         panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG] is methylated, wherein the group comprises at         least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and         denoted by [CG]; and     -   (b) diagnosing bladder cancer in the individual when at least 25         MVPs of the group of (a) are methylated.

The invention additionally provides a method of diagnosing bladder cancer in an individual comprising:

-   -   (a) obtaining data which identify whether each one of a group of         MVPs selected from a panel comprising the MVPs identified in SEQ         ID NOS 1 to 150 and denoted by [CG] is methylated, wherein the         group comprises at least 25 of the MVPs identified in SEQ ID NOS         1 to 150 and denoted by [CG]; and     -   (b) diagnosing bladder cancer in the individual when at least 25         MVPs of the group of (a) are methylated;         wherein the data were obtained by a method comprising:     -   i. obtaining DNA from the sample; and     -   ii. determining whether MVPs are methylated in the DNA.

In any of the methods described above the cancer may be a non-muscle invasive bladder cancer (NMIBC). The cancer may be a muscle invasive bladder cancer (MIBC).

The invention additionally provides an array capable of discriminating between methylated and non-methylated forms of MVPs; the array comprising oligonucleotide probes specific for a methylated form of each MVP in a MVP panel and oligonucleotide probes specific for a non-methylated form of each MVP in the panel; wherein the panel consists of at least 25 MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150.

In certain embodiments the array is not an Infinium HumanMethylation450 BeadChip array. In certain embodiments the number of MVP-specific oligonucleotide probes of the array is less than 482,421, preferably 482,000 or less, 480,000 or less, 450,000 or less, 440,000 or less, 430,000 or less, 420,000 or less, 410,000 or less, or 400,000 or less.

In an array as described above, the panel may consist of at least 40 MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150; preferably at least 50 MVPs, at least 60 MVPs, at least 70 MVPs, at least 80 MVPs, at least 90 MVPs, at least 100 MVPs, at least 110 MVPs, at least 120 MVPs, at least 130 MVPs, at least 140 MVPs, at least 145 MVPs, or all 150 MVPs identified in SEQ ID NOS 1 to 150.

In an array as described above, the panel may include the MVPs defined by SEQ ID NOS 1 to 3, or the MVPs defined by SEQ ID NOS 1 to 5, or the MVPs defined by SEQ ID NOS 1 to 10, or the MVPs defined by SEQ ID NOS 1 to 20, or the MVPs defined by SEQ ID NOS 1 to 30, or the MVPs defined by SEQ ID NOS 1 to 40, or the MVPs defined by SEQ ID NOS 1 to 50, or the MVPs defined by SEQ ID NOS 1 to 60, or the MVPs defined by SEQ ID NOS 1 to 70, or the MVPs defined by SEQ ID NOS 1 to 80, or the MVPs defined by SEQ ID NOS 1 to 90, or the MVPs defined by SEQ ID NOS 1 to 100, or the MVPs defined by SEQ ID NOS 1 to 100, or the MVPs defined by SEQ ID NOS 1 to 120, or the MVPs defined by SEQ ID NOS 1 to 130, or the MVPs defined by SEQ ID NOS 1 to 140, or the MVPs defined by SEQ ID NOS 1 to 150.

The panel may include all MVPs defined by SEQ ID NOS 1 to 150.

In array as described above, the array may further comprise one or more oligonucleotides comprising a MVP selected from any of the MVPs defined in SEQ ID NOS 1 to 150, wherein the one or more oligonucleotides are hybridized to corresponding oligonucleotide probes of the array.

The one or more oligonucleotides may comprise at least 20 MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150; preferably at least 50 MVPs, at least 60 MVPs, at least 70 MVPs, at least 80 MVPs, at least 90 MVPs, at least 100 MVPs, at least 110 MVPs, at least 120 MVPs, at least 130 MVPs, at least 140 MVPs, at least 145 MVPs, or all 150 MVPs identified in SEQ ID NOS 1 to 150.

The one or more oligonucleotides may comprise the MVPs defined by SEQ ID NOS 1 to 10, or the MVPs defined by SEQ ID NOS 1 to 20, or the MVPs defined by SEQ ID NOS 1 to 30, or the MVPs defined by SEQ ID NOS 1 to 40, or the MVPs defined by SEQ ID NOS 1 to 50, or the MVPs defined by SEQ ID NOS 1 to 60, or the MVPs defined by SEQ ID NOS 1 to 70, or the MVPs defined by SEQ ID NOS 1 to 80, or the MVPs defined by SEQ ID NOS 1 to 90, or the MVPs defined by SEQ ID NOS 1 to 100, or the MVPs defined by SEQ ID NOS 1 to 110, or the MVPs defined by SEQ ID NOS 1 to 120, or the MVPs defined by SEQ ID NOS 1 to 130, or the MVPs defined by SEQ ID NOS 1 to 140, or the MVPs defined by SEQ ID NOS 1 to 150.

The one or more oligonucleotides may comprise all MVPs defined by SEQ ID NOS 1 to 150.

Arrays as described above may be obtainable by hybridizing to an array as described above a group of oligonucleotides each comprising a different MVP selected from any of the MVPs defined in SEQ ID NOS 1 to 150, and wherein the group comprises at least 40 oligonucleotides.

In such a hybridized array, the group may comprise at least 50 oligonucleotides. The group may comprise at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 145, or at least 150 oligonucleotides.

In a hybridized array, the group may comprise at least 40 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 20, or wherein the group may comprise at least 50 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 50, or wherein the group may comprise at least 60 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 60, or wherein the group may comprise at least 70 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 70, or wherein the group may comprise at least 80 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 80, or wherein the group may comprise at least 90 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 90, or wherein the group may comprise at least 100 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 100, or wherein the group may comprise at least 110 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 110, or wherein the group may comprise at least 120 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 120, or wherein the group may comprise at least 130 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 130, or wherein the group may comprise at least 140 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 140, or wherein the group may comprise at least 145 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 145, or wherein the group may comprise at least 150 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 150. The group may comprise at least the 150 oligonucleotides comprising the MVPs defined by SEQ ID NOS 1 to 150.

The invention also provides a process for making the hybridized array as defined above, comprising contacting an array as defined above with a group of oligonucleotides each comprising a different MVP selected from any of the MVPs defined in SEQ ID NOS 1 to 150, and wherein the group comprises at least 25 oligonucleotides.

The invention also provides a process for making a hybridized array as defined above, comprising contacting an array as defined above with a group of oligonucleotides as defined above.

The invention also provides a kit comprising any of the arrays described above.

The kit may further comprise a DNA modifying regent that is capable of modifying a non-methylated cytosine in a MVP dinucleotide but is not capable of modifying a methylated cytosine in a MVP dinucleotide, optionally wherein the dinucleotide is CpG. The DNA modifying regent may be a bisulphite reagent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Heatmap of 9786 MVPs (1746 hypermethylated MVPs, 8040 hypomethylated MVPs) between bladder cancer (red) and 30 normal urothelium (blue).

FIG. 2. Heatmap of the 150 UroMark loci for non-cancer urine (n=10), normal urothelium (n=30), bladder cancer (n=81) and blood (n=489).

FIG. 3. Heat map of the 150 loci involved in the UroMark assay for normal urothelium (Blue) bladder cancer (Red) compared with the predicted (Light blue/light red) and actual (Blue/Red) status of bladder cancer.

FIG. 4. MDS plots for bladder tumour and normal urothelium based on the methylation state of 150 loci in bladder cancer samples from UCL and TCGA Bladder Cancer. The MDS (Multidimensional scaling) plot represents the dissimilarly of phenotypes based on the methylation state of the 150 loci with the panel, and clearly shows that the 150 marker can accurately separate tumour from normal bladder. Axis represent the Euclidean distance between samples.

FIG. 5. ROC plot for UroMark model for the detection of bladder cancer in urine.

FIG. 6. ROC plots for top performing (A) 3, (B) 5 and (C) 10 marker panels. Top 3 MVPs are listed as SEQ ID NOs: 1 to 3, Top 5 MVPs are listed as SEQ ID NOs: 1 to 5, Top 10 MVPs are listed as SEQ ID NOs: 1 to 10, all in rank order (see Table 1).

FIG. 7. ROC plot for UroMark model for the detection of bladder cancer from 176 unique urine samples (98 non-cancer urines and 78 cancer urines).

FIG. 8. Comparison of DNA quality (concentration, purity and integrity) from patients' urine. A) Concentration and purity of samples collected in clinic (n=123) compared with samples collected at home (n=41) where red indicates low yield or purity, amber indicates moderate yield and intermediate purity and green is high yield and purity. B) Percentage of samples exhibiting low, intermediate or high yield and purity. C) Representative Bioanalyzer electropherogram demonstrating recovery of high molecular weight of DNA in samples with concentrations quantifiable by spectrophotometry. D) Increased median yield and improved purity of urinary DNA using an extended digest step at 21° C. compared with the manufacturer's standard protocol of 56° C. for 1 hour. E) Comparison of two urine preservation methods: UCL established standard operating procedure versus Norgen urine preservation tubes. The number of bacteria were quantified by qPCR for the rpoB gene and expressed as copies rpoB per copy of human YWHAZ. Data are the mean+/− standard deviation.

FIG. 9. ROC plot for UroMark model for the detection of bladder cancer from Validation Cohort 2-96 unique urine samples (64 non-cancer urines and 32 cancer urines).

FIG. 10. ROC plot for UroMark model for the detection of bladder cancer from Validation Cohort 3-92 urine samples (65 non-cancer urines and 27 cancer urines).

DETAILED DESCRIPTION OF THE INVENTION

Bladder Cancer

As discussed above, bladder cancers represent one of the most prevalent groups of cancers in the western world. Transitional cell carcinoma is the most common type, and accounts for approximately 90% of bladder cancers. Transitional cell carcinomas arise from the transitional epithelium, which is a tissue lining the inner surface of the bladder. The remaining 10% of bladder cancers are mainly comprised of squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma. Squamous cell carcinoma also arises from epithelial tissue, from squamous cells. These are thin, flat cells found in the most superficial epithelial layer. Adenocarcinomas form from epithelial cells having glandular characteristics and/or origin. Sarcomas derive from cells of mesenchymal origin, such as the cells of the fat and muscle layers of the bladder. Small cell carcinomas have a rapid doubling time and are capable of earlier metastases, making them particularly aggressive.

Bladder cancers may also be classified as non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC).

The diagnostic and treatment methods described herein are capable of positively identifying malignant cells of all classifications of bladder cancer. Thus, any of the methods described herein may be used to diagnose transitional cell carcinoma of the bladder, squamous cell carcinoma of the bladder, adenocarcinoma of the bladder, sarcoma of the bladder, small cell carcinoma of the bladder, metastatic bladder cancer, leiomyosarcoma (a tumor arising from smooth muscle), lymphoma (a tumor that usually arises in the lymph nodes), malignant melanoma (a tumor that usually arises from the skin) and large cell neuroendocrine carcinoma. Primary forms and recurrent forms of bladder cancer are included. The cancer to be diagnosed or treated as described herein may be a urothelial cell cancer. Thus, the cancer may be cancer of the ureter, urethra or renal pelvis.

The most preferred patient type to which the diagnostic assays described herein are applicable are humans. The diagnostic assays described herein may also be used to identify bladder cancer in a non-human animal. For example, non-human animals may contain tissue derived from humans, e.g. xenografts. Thus, diagnostic assays may be used to diagnose human bladder cancer in an animal model of human bladder cancer. Typical non-human animals to which the diagnostic assays described herein are applicable are rodents such as rats or mice.

Methylation Variable Positions (MVPs)

Methylation of DNA is a recognised form of epigenetic modification which has the capability of altering the expression of genes and other elements such as microRNAs [51]. In cancer development and progression, methylation may have the effect of e.g. silencing tumor suppressor genes and/or increasing the expression of oncogenes. Other forms of dysregulation may occur as a result of methylation. Methylation of DNA occurs at discrete loci which are predominately dinucleotide consisting of a CpG motif, but may also occur at CHH motifs (where H is A, C, or T). During methylation, a methyl group is added to the fifth carbon of cytosine bases to create methylcytosine.

Methylation can occur throughout the genome and is not limited to regions with respect to an expressed sequence such as a gene. Methylation typically, but not always, occurs in a promoter or other regulatory region of an expressed sequence.

A Methylation Variable Position (MVP) as defined herein is any dinucleotide locus which may show a variation in its methylation status between phenotypes, i.e. between tumour and normal tissue. An MVP is preferably a CpG or a CHH dinucleotide motif. An MPV as defined herein is not limited to the position of the locus with respect to a corresponding expressed sequence.

Typically, an assessment of DNA methylation status involves analysing the presence or absence of methyl groups in DNA, for example methyl groups on the 5^(th) position of one or more cytosine nucleotides. Preferably, the methylation status of one or more cytosine nucleotides present as a CpG dinucleotide (where C stands for Cytosine, G for Guanine and p for the phosphate group linking the two) is assessed.

By assessing the methylation status of an MVP or determining whether an MVP is methylated it is meant that a determination is made as to whether an MVP was methylated or unmethylated in the starting sample of DNA obtained from the individual prior to subsequent processing.

An MVP is herein defined as methylated if one or more alleles of that MVP in a sample of genomic DNA from the patient is determined to possess one or more methylated CpG dinucleotide loci.

In any of the methods described herein, the MVPs determined to be methylated are methylated relative to normal urothelium control and/or whole blood control.

Specific MVPs useful for diagnostic purposes are set forth in Table 1 and are identified by SEQ ID number, as well as Illumina ID number (Ilmn ID). Exemplary primers for amplifying the defined MVPs are set forth in Table 2 and are also identified by SEQ ID number.

Identification and Assessment of Methylation Variable Position (MVP) Status

A variety of techniques are available for the identification and assessment of Methylation Variable Positions (MVPs), as will be outlined briefly below. The diagnostic methods described herein encompass any suitable technique for the determination of MVP status.

Methyl groups are lost from a starting DNA molecule during conventional in vitro handling steps such as PCR. To avoid this, techniques for the detection of methyl groups commonly involve the preliminary treatment of DNA prior to subsequent processing, in a way that preserves the methylation status information of the original DNA molecule. Such preliminary techniques involve three main categories of processing, i.e. bisulphite modification, restriction enzyme digestion and affinity-based analysis. Products of these techniques can then be coupled with sequencing or array-based platforms for subsequent identification or qualitative assessment of MVP methylation status.

Techniques involving bisulphite modification of DNA have become the most common methods for detection and assessment of methylation status of CpG dinucleotide. Treatment of DNA with bisulphite, e.g. sodium bisulphite, converts cytosine bases to uracil bases, but has no effect on 5-methylcytosines. Thus, the presence of a cytosine in bisulphite-treated DNA is indicative of the presence of a cytosine base which was previously methylated in the starting DNA molecule. Such cytosine bases can be detected by a variety of techniques. For example, primers specific for unmethylated versus methylated DNA can be generated and used for PCR-based identification of methylated CpG dinucleotides. A separation/capture step may be performed, e.g. using binding molecules such as complementary oligonucleotide sequences. Standard and next-generation DNA sequencing protocols can also be used.

In other approaches, methylation-sensitive enzymes can be employed which digest or cut only in the presence of methylated DNA. Analysis of resulting fragments is commonly carried out using microarrays.

Affinity-based techniques exploit binding interactions to capture fragments of methylated DNA for the purposes of enrichment. Binding molecules such as anti-5-methylcytosine antibodies are commonly employed prior to subsequent processing steps such as PCR and sequencing.

Olkhov-Mitsel and Bapat (2012) [51] provide a comprehensive review of techniques available for the identification and assessment of MVP-based biomarkers involving methylcytosine.

For the purposes of assessing the methylation status of the MVP-based biomarkers characterised and described herein, any suitable method can be employed.

Preferred methods involve bisulphite treatment of DNA, including amplification of the identified MVP loci for methylation specific PCR and/or sequencing and/or assessment of the methylation status of target loci using methylation-discriminatory microarrays.

Amplification of MVP loci can be achieved by a variety of approaches. Preferably, MVP loci are amplified using PCR. MVP may also be amplified by other techniques such as multiplex ligation-dependent probe amplification (MLPA). A variety of PCR-based approaches may be used. For example, methylation-specific primers may be hybridized to DNA containing the MVP sequence of interest. Such primers may be designed to anneal to a sequence derived from either a methylated or non-methylated MVP locus. Following annealing, a PCR reaction is performed and the presence of a subsequent PCR product indicates the presence of an annealed MVP of identifiable sequence. In such methods, DNA is bisulphite converted prior to amplification. Such techniques are commonly referred to as methylation specific PCR (MSP) [53].

In other techniques, PCR primers may anneal to the MVP sequence of interest independently of the methylation status, and further processing steps may be used to determine the status of the MVP. Assays are designed so that the MVP site(s) are located between primer annealing sites. This method scheme is used in techniques such as bisulphite genomic sequencing [54], COBRA [55], Ms-SNuPE [56]. In such methods, DNA can be bisulphite converted before or after amplification.

Preferably, small-scale PCR approaches are used. Such approaches commonly involve mass partitioning of samples (e.g. digital PCR). These techniques offer robust accuracy and sensitivity in the context of a highly miniaturised system (pico-liter sized droplets), ideal for the subsequent handling of small quantities of DNA obtainable from the potentially small volume of cellular material present in biological samples, particularly urine samples. A variety of such small-scale PCR techniques are widely available. For example, microdroplet-based PCR instruments are available from a variety of suppliers, including RainDance Technologies, Inc. (Billerica, Mass.; see the RainDance Technologies, Inc. website on the world wide web) and Bio-Rad, Inc. (see the Bio-Rad Inc. website on the world wide web). Microarray platforms may also be used to carry out small-scale PCR. Such platforms may include microfluidic network-based arrays e.g. available from Fluidigm Corp. (see the Fluidigm Corp. website on the world wide web).

Following amplification of MVP loci, amplified PCR products may be coupled to subsequent analytical platforms in order to determine the methylation status of the MVPs of interest. For example, the PCR products may be directly sequenced to determine the presence or absence of a methylcytosine at the target MVP or analysed by array-based techniques.

Any suitable sequencing techniques may be employed to determine the sequence of target DNA. In the methods of the present invention the use of high-throughput, so-called “second generation”, “third generation” and “next generation” techniques to sequence bisulphite-treated DNA are preferred.

In second generation techniques, large numbers of DNA molecules are sequenced in parallel. Typically, tens of thousands of molecules are anchored to a given location at high density and sequences are determined in a process dependent upon DNA synthesis. Reactions generally consist of successive reagent delivery and washing steps, e.g. to allow the incorporation of reversible labelled terminator bases, and scanning steps to determine the order of base incorporation. Array-based systems of this type are available commercially e.g. from Illumina, Inc. (San Diego, Calif.; see the IIlumina, Inc. website on the world wide web).

Third generation techniques are typically defined by the absence of a requirement to halt the sequencing process between detection steps and can therefore be viewed as real-time systems. For example, the base-specific release of hydrogen ions, which occurs during the incorporation process, can be detected in the context of microwell systems (e.g. see the Ion Torrent system available from Life Technologies; see the Life Technologies website on the world wide web). Similarly, in pyrosequencing the base-specific release of pyrophosphate (PPi) is detected and analysed. In nanopore technologies, DNA molecules are passed through or positioned next to nanopores, and the identities of individual bases are determined following movement of the DNA molecule relative to the nanopore. Systems of this type are available commercially e.g. from Oxford Nanopore (see the Oxford Nanopore website on the world wide web). In an alternative method, a DNA polymerase enzyme is confined in a “zero-mode waveguide” and the identity of incorporated bases are determined with florescence detection of gamma-labeled phosphonucleotides (see e.g. Pacific Biosciences; see the Pacific Biosciences website on the world wide web).

In other methods in accordance with the invention sequencing steps may be omitted. For example, amplified PCR products may be applied directly to hybridization arrays based on the principle of the annealing of two complementary nucleic acid strands to form a double-stranded molecule. Hybridization arrays may be designed to include probes which are able to hybridize to amplification products of an MVP and allow discrimination between methylated and non-methylated loci. For example, probes may be designed which are able to selectively hybridize to an MVP locus containing thymine, indicating the generation of uracil following bisulphite conversion of an unmethylated cytosine in the starting template DNA. Conversely, probes may be designed which are able to selectively hybridize to an MVP locus containing cytosine, indicating the absence of uracil conversion following bisulphite treatment. This corresponds with a methylated MVP locus in the starting template DNA.

Following the application of a suitable detection system to the array, computer-based analytical techniques can be used to determine the methylation status of an MVP. Detection systems may include, e.g. the addition of fluorescent molecules following a methylation status-specific probe extension reaction. Such techniques allow MVP status determination without the specific need for the sequencing of MVP amplification products. Such array-based discriminatory probes may be termed methylation-specific probes.

Any suitable methylation-discriminatory microarrays may be employed to assess the methylation status of the MVPs described herein. A preferred methylation-discriminatory microarray system is provided by Illumina, Inc. (San Diego, Calif.; see the Illumina, Inc. website on the world wide web). In particular, the Infinium HumanMethylation450 BeadChip array system may be used to assess the methylation status of diagnostic MVPs for bladder cancer as described herein. Such a system exploits the chemical modifications made to DNA following bisulphite treatment of the starting DNA molecule. Briefly, the array comprises beads to which are coupled oligonucleotide probes specific for DNA sequences corresponding to the unmethylated form of an MVP, as well as separate beads to which are coupled oligonucleotide probes specific for DNA sequences corresponding to the methylated form of an MVP. Candidate DNA molecules are applied to the array and selectively hybridize, under appropriate conditions, to the oligonucleotide probe corresponding to the relevant epigenetic form. Thus, a DNA molecule derived from an MVP which was methylated in the corresponding genomic DNA will selectively attach to the bead comprising the methylation-specific oligonucleotide probe, but will fail to attach to the bead comprising the non-methylation-specific oligonucleotide probe. Single-base extension of only the hybridized probes incorporates a labeled ddNTP, which is subsequently stained with a fluorescence reagent and imaged. The methylation status of the MVP may be determined by calculating the ratio of the fluorescent signal derived from the methylated and unmethylated sites.

Because the bladder cancer-specific diagnostic MVP biomarkers defined herein were initially identified using the Illumina Infinium HumanMethylation450 BeadChip array system, the same chip system can be used to interrogate those same MVPs in the diagnostic assays described herein. Alternative or customised arrays could, however, be employed to interrogate the bladder cancer-specific diagnostic MVP biomarkers defined herein, provided that they comprise means for interrogating all MVPs for a given method, as defined herein.

Techniques involving combinations of the above-described methods may also be used. For example, DNA containing MVP sequences of interest may be hybridized to microarrays and then subjected to DNA sequencing to determine the status of the MVP as described above.

In the methods described above, sequences corresponding to MVP loci may also be subjected to an enrichment process. DNA containing MVP sequences of interest may be captured by binding molecules such as oligonucleotide probes complementary to the MVP target sequence of interest. Sequences corresponding to MVP loci may be captured before or after bisulphite conversion or before or after amplification. Probes may be designed to be complementary to bisulphite converted DNA. Captured DNA may then be subjected to further processing steps to determine the status of the MVP, such as DNA sequencing steps.

Capture/separation steps may be custom designed. Alternatively a variety of such techniques are available commercially, e.g. the SureSelect target enrichment system available from Agilent Technologies (see the Agilent Technologies website on the world wide web). In this system biotinylated “bait” or “probe” sequences (e.g. RNA) complementary to the DNA containing MVP sequences of interest are hybridized to sample nucleic acids. Streptavidin-coated magnetic beads are then used to capture sequences of interest hybridized to bait sequences. Unbound fractions are discarded. Bait sequences are then removed (e.g. by digestion of RNA) thus providing an enriched pool of MVP target sequences separated from non-MVP sequences. In a preferred method of the invention, template DNA is subjected to bisulphite conversion and target loci are then amplified by small-scale PCR such as microdroplet PCR using primers which are independent of the methylation status of the MVP. Following amplification, samples are subjected to a capture step to enrich for PCR products containing the target MVP, e.g. captured and purified using magnetic beads, as described above. Following capture, a standard PCR reaction is carried out to incorporate DNA sequencing barcodes into MVP-containing amplicons. PCR products are again purified and then subjected to DNA sequencing and analysis to determine the presence or absence of a methylcytosine at the target genomic MVP [31].

The MVP biomarker loci defined herein are identified e.g. by Illumina® identifiers (IlmnID) These MVP loci identifiers refer to individual MVP sites used in the commercially available Illumina® Infinium Human Methylation450 BeadChip kit. The identity of each MVP site represented by each MVP loci identifier is publicly available from the Illumina, Inc. website under reference to the MVP sites used in the Infinium Human Methylation450 BeadChip kit.

Further information regarding MVP loci identification used in Illumina, Inc products is found in the technical note entitled “Technical Note: Epigenetics. CpG Loci Identification. A guide to Illumina's method for unambiguous CpG loci identification and tracking for the Golden Gate® and Infinium® Assay for Methylation” published in 2010 and found at

the Illumina, Inc. website on the world wide web.

Further information regarding the Illumina® Infinium Human Methylation450 BeadChip system can be found at:

the Illumina, Inc. website on the world wide web;

To complement evolving public databases to provide accurate MVP/CpG loci identifiers and strand orientation, Illumina® has developed a method to consistently designate MVP/CpG loci based on the actual or contextual sequence of each individual MVP/CpG locus. To unambiguously refer to MVP/CpG loci in any species, Illumina® has developed a consistent and deterministic MVP loci database to ensure uniformity in the reporting of methylation data. The Illumina® method takes advantage of sequences flanking a MVP locus to generate a unique MVP locus cluster ID. This number is based on sequence information only and is unaffected by genome version. Illumina's standardized nomenclature also parallels the TOP/BOT strand nomenclature (which indicates the strand orientation) commonly used for single nucleotide polymorphism (SNP) designation.

Illumina® Identifiers for the Infinium Human Methylation450 BeadChip system are also available from public repositories such as Gene Expression Omnibus (GEO) (see the GEO webpage on the National Center for Biotechnology Information website on the world wide web).

An MVP as defined herein thus refers to the CG dinucleotide motif identified in relation to each SEQ ID NO. and Illumina Identifier (Ilmn ID) as listed in Table 1, wherein the cytosine base of the dinucleotide (noted in bold and square brackets in the sequences listed at Table 1) may (or may not) be modified. Thus by determining the methylation status of a CpG defined by or identified in a given SEQ ID NO., or determining whether such a CpG is methylated, it is meant that a determination is made as to whether the cytosine of the CG dinucleotide motif identified in bold and in square brackets in a sequence shown in Table 1 is methylated or not at one or more loci in the sample of DNA from the individual, accepting that variation in the sequence upstream and downstream of any given CpG may exist due to sequencing errors or variation between individuals.

The invention provides a method of diagnosing bladder cancer in an individual comprising:

(a) providing DNA from a sample from the individual;

(b) determining whether each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] is methylated, wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]; and

(c) diagnosing bladder cancer in the individual when at least 25 of the MVPs of the group of (b) are methylated.

In any such method described herein, the group of MVPs (i.e. those MVPs the methylation status of which are to be determined) may comprise 26 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]; or the group may comprise 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or more, 61 or more, 62 or more, 63 or more, 64 or more, 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, 70 or more, 71 or more, 72 or more, 73 or more, 74 or more, 75 or more, 76 or more, 77 or more, 78 or more, 79 or more, 80 or more, 81 or more, 82 or more, 83 or more, 84 or more, 85 or more, 86 or more, 87 or more, 88 or more, 89 or more, 90 or more, 91 or more, 92 or more, 93 or more, 94 or more, 95 or more, 96 or more, 97 or more, 98 or more, 99 or more, 100 or more, 101 or more, 102 or more, 103 or more, 104 or more, 105 or more, 106 or more, 107 or more, 108 or more, 109 or more, 110 or more, 111 or more, 112 or more, 113 or more, 114 or more, 115 or more, 116 or more, 117 or more, 118 or more, 119 or more, 120 or more, 121 or more, 122 or more, 123 or more, 124 or more, 125 or more, 126 or more, 127 or more, 128 or more, 129 or more, 130 or more, 131 or more, 132 or more, 133 or more, 134 or more, 135 or more, 136 or more, 137 or more, 138 or more, 139 or more, 140 or more, 141 or more, 142 or more, 143 or more, 144 or more, 145 or more, 146 or more, 147 or more or 148 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]. The group may comprise 149 or 150 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

In any of the methods described above, bladder cancer may be diagnosed when at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated. Bladder cancer may be diagnosed when 26 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated; or when 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or more, 61 or more, 62 or more, 63 or more, 64 or more, 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, 70 or more, 71 or more, 72 or more, 73 or more, 74 or more, 75 or more, 76 or more, 77 or more, 78 or more, 79 or more, 80 or more, 81 or more, 82 or more, 83 or more, 84 or more, 85 or more, 86 or more, 87 or more, 88 or more, 89 or more, 90 or more, 91 or more, 92 or more, 93 or more, 94 or more, 95 or more, 96 or more, 97 or more, 98 or more, 99 or more, 100 or more, 101 or more, 102 or more, 103 or more, 104 or more, 105 or more, 106 or more, 107 or more, 108 or more, 109 or more, 110 or more, 111 or more, 112 or more, 113 or more, 114 or more, 115 or more, 116 or more, 117 or more, 118 or more, 119 or more, 120 or more, 121 or more, 122 or more, 123 or more, 124 or more, 125 or more, 126 or more, 127 or more, 128 or more, 129 or more, 130 or more, 131 or more, 132 or more, 133 or more, 134 or more, 135 or more, 136 or more, 137 or more, 138 or more, 139 or more, 140 or more, 141 or more, 142 or more, 143 or more, 144 or more, 145 or more, 146 or more, 147 or more, or 148 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated. Bladder cancer may be diagnosed when 149 or 150 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated.

Preferably, bladder cancer may be diagnosed when 40 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated.

Bladder cancer may also be diagnosed when 50 or more, 60 or more, 70 or more or 80 or more, 90 or more or 100 or more of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated.

In any of the methods described above the MVPs determined to be methylated may include the MVPs identified in SEQ ID NOS 1 to 3 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 5 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 10 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 20 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 30 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 40 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 50 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 60 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 70 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 80 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 90 and denoted by [CG], or may include the MVPs identified in SEQ ID NOS 1 to 100 and denoted by [CG].

In one embodiment, the group of MVPs (i.e. those MVPs the methylation status of which are to be determined) may comprises all 150 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], and in this method bladder cancer is diagnosed in the individual when at least 40 of the MVPs selected from the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are methylated, and in this method the MVPs determined to be methylated include the MVPs identified in SEQ ID NOS 1 to 10 and denoted by [CG].

Bioinformatic Tools and Statistical Metrics

Software programs which aid in the in silico analysis of bisulphite converted DNA sequences and in primer design for the purposes of methylation-specific analyses are generally available and have been described previously [57, 58, 59].

Sensitivity and specificity metrics for bladder cancer diagnosis based on the MVP methylation status assays described herein may be defined using standard receiver operating characteristic (ROC) statistical analysis [52]. In ROC analysis 100% sensitivity corresponds to a finding of no false negatives, and 100% specificity corresponds to a finding of no false positives.

Based on analyses conducted using a panel of 150 MVP biomarkers, a bladder cancer diagnostic assay in accordance with the invention described herein can achieve a ROC sensitivity of 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99%. The ROC sensitivity may be 100%.

Diagnostic assays in accordance with the invention can achieve a ROC specificity of 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99%. The ROC specificity may be 100%.

Diagnostic assays in accordance with the invention may have an associated combination of ROC sensitivity and ROC specificity values wherein the combination is any one of the above-listed sensitivity values and any one of the above-listed specificity values, provided that the sensitivity value is equal to or less than the specificity value.

Thus, the ROC specificity may be 100% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or 100%.

The ROC specificity may be 99% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or 99%.

The ROC specificity may be 98% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or 98%.

The ROC specificity may be 97% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or 97%.

The ROC specificity may be 96% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or 96%.

The ROC specificity may be 95% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or 95%.

The ROC specificity may be 94% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or greater, 93% or 94%.

The ROC specificity may be 93% and the ROC sensitivity may be 90% or greater, 91% or greater, 92% or 93%.

The ROC specificity may be 92% and the ROC sensitivity may be 90% or greater, 91% or 92%.

The ROC specificity may be 91% and the ROC sensitivity may be 90% or 91%.

The ROC specificity may be 90% and the ROC sensitivity may be 90%. Preferably, the assay may achieve a ROC sensitivity of 95% or greater and a ROC specificity of 90% or greater; preferably a ROC sensitivity of 96% and a ROC specificity of 97%.

ROC plots corresponding to example methods representative of the diagnostic methods defined herein are presented at FIGS. 5 and 7, demonstrating the exquisite sensitivity and selectivity of the MVP-based assays. This contrasts with assays conducted with smaller panels of biomarkers, which have been described previously. Thus, comparative data demonstrate the superior predictive power of the assays defined herein.

A further metric which can be employed to classify the accuracy of the MVP-based assays is ROC AUC. In ROC analysis, the area under the curve of a ROC plot (AUC) is a metric for binary classification. In a random binary classifier the number of true positives and false positives will be approximately equal. In this situation the AUC score for the ROC plot will be 0.5. In a perfect binary classifier the number of true positives will be 100% and the number of false positives will be 0%. In this situation the AUC score for the ROC plot will be 1.

Based on analyses conducted using biomarkers described herein, a bladder cancer diagnostic assay in accordance with the invention can achieve a ROC AUC of 0.90 or greater, 0.91 or greater, 0.92 or greater, 0.93 or greater, 0.94 or greater, 0.95 or greater, 0.96 or greater, 0.97 or greater, 0.98 or greater, 0.99 or 1. Preferably the diagnostic assay can achieve a ROC AUC of 0.98 or greater.

Bladder cancer diagnostic tests based on the MVP methylation status assays described herein may also be characterised using a Negative Predictive Value (NPV) metric. The NPV is a measure of the proportion of negative results that are true negative results.

Based on analyses conducted using a panel of 150 MVP biomarkers, a bladder cancer diagnostic assay in accordance with the invention described herein can achieve an NPV of 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or 100%.

Biological Samples

The bladder cancer diagnostic assays described herein may be performed on any suitable biological material obtained from the patient. Preferred biological material is urine. However, samples of bladder tissue, e.g. obtained via biopsy or aspirates, or obtained from preserved samples (e.g. cryopreserved material, tissue sections etc.) may be used. Samples of biological material may also include solid tissue samples, aspirates, samples of biological fluids, blood, serum, plasma, ascitic fluid, lymph, peripheral blood, cerebrospinal fluid, fine needle aspirate, saliva, sputum, bone marrow, skin, epithelial samples (including buccal, cervical or vaginal epithelia) or other tissue derived from the ectoderm, vaginal fluid, semen etc. Tissue scrapes may include biological material from e.g. buccal, oesophageal, bladder, vaginal, urethral or cervical scrapes. The cells of the sample may comprise inflammatory cells, such as lymphocytes.

Any of the assays and methods described herein may involve providing a biological sample from the patient as the source of patient DNA for methylation analysis.

Any of the assays and methods described herein may involve obtaining patient DNA from a biological sample which has previously been obtained from the patient.

Any of the assays and methods described herein may involve obtaining a biological sample from the patient as the source of patient DNA for methylation analysis. Procedures for obtaining a biological sample from the patient may be non-invasive, such as collecting cells from urine. Alternatively, invasive procedures such as biopsies may be used.

In the methods described herein the level of detection is such that 2 tumor cells may be detected in a sample comprising 150,000 cells or more. In such methods the sample may comprise 160,000 cells or more, 170,000 cells or more, 180,000 cells or more, 190,000 cells or more, 200,000 cells or more, 210,000 cells or more, 220,000 cells or more, 230,000 cells or more, 240,000 cells or more, 250,000 cells or more, 260,000 cells or more, 270,000 cells or more, 280,000 cells or more, 280,000 cells or more, or 300,000 cells or more.

In any such method, the number of tumor cells that can be detected is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 20000 or more, 30000 or more, 40000 or more, 50000 or more, 60000 or more, 70000 or more, 80000 or more, 90000 or more or 100000 or more.

Methods of Treatment

The invention also encompasses the performance of one or more treatment steps following a positive diagnosis of bladder cancer by the diagnostic methods described herein.

Thus the invention also encompasses a method of treating bladder cancer in an individual comprising:

(a) obtaining DNA from a sample from the individual and determining whether each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] is methylated, wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG];

(b) diagnosing bladder cancer in the individual when at least 25 MVPs of the group of (a) are methylated; and

(c) administering one or more bladder cancer treatments to the individual.

The invention also encompasses a method of treating bladder cancer in an individual comprising:

(a) providing DNA from a sample from the individual and determining whether each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] is methylated, wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG];

(b) diagnosing bladder cancer in the individual when at least 25 MVPs of the group of (a) are methylated; and

(c) administering one or more bladder cancer treatments to the individual.

The invention also encompasses a method of treating bladder cancer in an individual comprising:

(a) determining whether each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] is methylated in DNA from a sample from the individual, wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG];

(b) diagnosing bladder cancer in the individual when at least 25 MVPs of the group of (a) are methylated; and

(c) administering one or more bladder cancer treatments to the individual.

The invention also encompasses a method of treating bladder cancer in an individual comprising administering one or more bladder cancer treatments to the individual, wherein the individual has been diagnosed with bladder cancer by steps comprising:

(a) providing DNA from a sample from the individual and determining whether each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] is methylated, wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]; and

(b) diagnosing bladder cancer in the individual when at least 25 MVPs of the group of (a) are methylated.

In any of the above-described methods of treating bladder cancer, the group of MVPs which are selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] (i.e. the group of MVPs whose methylation status is to be determined) may comprise any number of MVPs as described and defined herein, provided that the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]. In any of these methods, bladder cancer may be diagnosed in the individual when the number of MVPs of the group which are determined to be methylated is any number of MVPs as described and defined herein, provided that at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] are determined to be methylated.

Thus, the invention encompasses administration of one or more surgical procedures, one or more chemotherapeutic agents, one or more immunotherapeutic agents, one or more radiotherapeutic agents, one or more hormonal therapeutic agents or any combination of the above following a positive diagnosis of bladder cancer.

Surgical procedures include transurethral resection of bladder tumor (TURBT), cystectomy, open radical cystectomy (ORC), laparoscopic radical cystectomy (LRC) and robot-assisted radical cystectomy (RARC).

Chemotherapeutic agents include the following. Alkylating agents, which include the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and platinum based derivatives, as well as the non-classical alkylating agents. Antimetabolites, which include the anti-folates, fluoropyrimidines, deoxynucleoside analogues and thiopurines. Microtubule disrupting agents, which include the vinca alkaloids and taxanes, as well as dolastatin 10 and derivatives thereof. Topoisomerase inhibitors, which include camptothecin, irinotecan and topotecan. Topoisomerase II poisons, which include etoposide, doxorubicin, mitoxantrone and teniposide. Topoisomerase II catalytic inhibitors, which include novobiocin, merbarone, and aclarubicin. Cytotoxic antibiotics, which include anthracyclines, actinomycin, bleomycin, plicamycin, and mitomycin.

Combinations of agents include but are not limited to MVAC (Methotrexate, Vinblastine, Vinblastine and Vinblastine), Gem-Cis (GC) (Gemcitabine and Cisplatin), Lapatinib and gemcitabine.

Immunotherapeutics include bacilli Calmette-Guérin (BCG) immunotherapy as well as monoclonal antibodies and antibody-drug conjugates. Antibody-drug conjugates include antibodies conjugated to microtubule disrupting agents and DNA modifying agents as described above.

Combination therapies include intravesical, sequential BCG, followed by electromotive administration (EMDA) of MMC (EMDA-MMC) as well as microwave-induced bladder wall hyperthermia (HT) and intravesical MMC.

Cancer therapeutic agents are administered to a subject already suffering from a disorder or condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for a given purpose will depend on the severity of the disease as well as the weight and general state of the subject. As used herein, the term “subject” includes any human.

Arrays

The invention also encompasses arrays capable of discriminating between methylated and non-methylated forms of MVPs as defined herein; the arrays may comprise oligonucleotide probes specific for methylated forms of MVPs as defined herein and oligonucleotide probes specific for non-methylated forms of MVPs as defined herein.

By “specific” it is meant that the probes comprise sequences which are complementary to those of the oligonucleotides comprising the MVP such they may hybridize, particularly under stringent conditions.

In some embodiments the array is not an Illumina Infinium HumanMethylation450 BeadChip array (Infinium HumanMethylation450 BeadChip array).

Separately or additionally, in some embodiments the number of MVP-specific oligonucleotide probes of the array is less than 482,421, preferably 482,000 or less, 480,000 or less, 450,000 or less, 440,000 or less, 430,000 or less, 420,000 or less, 410,000 or less, or 400,000 or less, 375,000 or less, 350,000 or less, 325,000 or less, 300,000 or less, 275,000 or less, 250,000 or less, 225,000 or less, 200,000 or less, 175,000 or less, 150,000 or less, 125,000 or less, 100,000 or less, 75,000 or less, 50,000 or less, 45,000 or less, 40,000 or less, 35,000 or less, 30,000 or less, 25,000 or less, 20,000 or less, 15,000 or less, 10,000 or less, 5,000 or less, 4,000 or less, 3,000 or less or 2,000 or less.

The invention further encompasses the use of any of the arrays as defined herein in any of the methods which require determining the methylation status of MVPs for the purposes of diagnosing bladder cancer cells in an individual.

Kits

Any of the arrays as defined herein may be comprised in a kit.

The kit may comprise any array as defined herein.

The kit may comprise any array as defined herein together with instructions for use.

The kit may additionally comprise a DNA modifying regent, such as a bisulphite reagent.

The kit may additionally comprise reagents for amplifying DNA, such as primers directed to any of the MVPs as defined herein as identified in SEQ ID NOS 1 to 150 (see Table 2).

Methods of Determining a Methylation Profile of a Sample

The invention further encompasses a method of determining a methylation profile of a sample from an individual, the method comprising:

-   -   i. providing DNA from a sample from the individual;     -   ii. determining whether each one of a group of MVPs selected         from a panel comprising the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG] is methylated, wherein the group         comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG]; and     -   iii. based on the methylation status of the MVPs of the group,         determining a methylation profile of the sample.

In any of the above-described methods of determining a methylation profile of a sample, the group of MVPs which are selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] (i.e. the group of MVPs whose methylation status is to be determined) may comprise any number of MVPs as described and defined herein, provided that the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

Furthermore, in any such methods, the methylation status of MVPs may be determined using any of the arrays described herein.

Further Methods

In any of the diagnostic methods described herein, the step of diagnosing bladder cancer in the individual may further comprise:

-   -   I. stratifying the grade of the tumor; and/or     -   II. determining the risk of recurrence of the tumor; and/or     -   III. determining the risk of progression of non-muscular         invasive disease; and/or     -   IV. determining the likely response to treatment therapy.

The invention also encompasses a method of determining the risk of the development of bladder cancer in an individual, the method comprising:

-   -   (a) providing DNA from a sample from the individual;     -   (b) determining whether each one of a group of MVPs selected         from a panel comprising the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG] is methylated, wherein the group         comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to         150 and denoted by [CG]; and     -   (c) based on the methylation status of the MVPs of the group,         determining the risk of the development of bladder cancer in the         individual.

In any such method of determining the risk of the development of bladder cancer in an individual, the group of MVPs which are selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG] (i.e. the group of MVPs whose methylation status is to be determined) may comprise any number of MVPs as described and defined herein, provided that the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

Furthermore, in any such methods, the methylation status of MVPs may be determined using any of the arrays described herein.

Further Uses

The invention also encompasses the use of a group of MVPs in the diagnosis of bladder cancer in an individual or in determining the risk of the development of bladder cancer in an individual.

In any such use, the group of MVPs are selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

In any such use, the group of MVPs may comprise any number of MVPs as described and defined herein, provided that the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].

In any of the above-described uses, the diagnosis of bladder cancer in an individual or the determination of the risk of the development of bladder cancer in an individual may be performed by any of the respective methods described and defined herein. Furthermore, in any such methods, the methylation status of MVPs may be determined using any of the arrays described herein.

The invention is illustrated by the following Examples:

EXAMPLES

Materials and Methods

Introduction

Emerging techniques that utilize next-generation DNA sequencing platforms hold particular promise for the development of highly sensitive epigenetic biomarker panels. For example, the microdroplet-based PCR amplification of bisulphite converted DNA, followed by next-generation sequencing of the amplified target loci developed by RainDance Technologies [30] enables the sensitive, specific and simultaneous amplification of up to 20,000 bisulfite-converted target loci. Highly parallel microdroplet-based PCR amplification of bisulphite-converted DNA has shown utility in the validation of epigenetic alterations in a range of tissues [31-33]. However, as yet it has not been applied to the development of non-invasive diagnostic biomarkers for the detection of bladder cancer.

To derive a sensitive assay for the detection of bladder cancer, the inventors have performed one of the largest independent studies of genome-wide methylation in bladder cancer to date. From this, a panel of bladder-specific epigenetic biomarkers have been defined and the sensitivity and specificity of a 150 loci panel using RainDrop-BS [31] on urinary DNA have been validated for the detection of bladder cancer with a high degree of diagnostic precision.

Study Population

Genome-wide DNA methylation profiling was performed on DNA from 81 bladder cancers and 30 age-matched normal urothelium samples collected from UCLH (London) Department of Urology and CIEMAT (Madrid). The cohort included 35 low-grade non-muscle-invasive cancers. Pathological review of representative H&E sections was conducted to include specimens with tumor cellularity >80%. Blood methylome data was download from the MARMAL-aid database ([34]; see the Marmal-aid website on the world wide web).

Independent validation data were obtained from the Cancer Genome Atlas Project (see The Cancer Genome Atlas Project at the National Cancer Institute website on the world wide web), consisting of MIBC bladder cancer and 20 normal urothelium samples.

For validation studies sequential urine samples were collected from patients attending UCLH one-stop haematuria and surveillance cystoscopy clinics (n=86 bladder cancer, n=96 non-cancer controls).

Urine Collection

Urine samples were obtained from patients attending haematuria clinics or undergoing cystoscopy for recurrent bladder cancer at University College Hospital. For comparison of clinic versus home urine samples, patients were asked to supply three samples: a clinic sample and two home samples, one of which should be a first void. 40-100 ml were obtained per sample. The home urine kit for one sample comprised up to four 25 ml sterile tubes, mailing tubes with absorbent pads and a pre-addressed padded envelope, designed to fit through a Royal Mail post box.

DNA Extraction and Quantification

Urinary DNA was extracted using a DNeasy blood and tissue kit (Qiagen, UK) in accordance with the manufacturer's instructions. DNA was quantified by spectrophotometry (Nanodrop 1000) and fluorometry (Qubit ds DNA HS assay, Invitrogen, UK).

RainDance Microdroplet PCR

For microdroplet PCR, 7.20 μl of bisulfite-treated (and optionally, whole-genome amplified) DNA were added to 4.70 μl of 10× High-Fidelity Buffer (Invitrogen), 1.80 μl of 50 mM MgSO₄ (Invitrogen), 1.62 μl of 10 mM dNTP solution mix (NEB), 3.60 μl of 4 M betaine solution (Sigma-Aldrich), 3.60 μl of droplet stabilizer (RainDance Technologies), 1.80 μl of 100% dimethyl sulfoxide (Sigma-Aldrich) and 0.72 μl of 5 U/μl Platinum Taq Polymerase High-Fidelity (Invitrogen), to a total volume of 25 μl. The sample plate was sealed using an ALPS 50V microplate heat sealer (Thermo Scientific).

The bisulfite-treated genomic DNA template mix was then applied to a fully automated ThunderStorm system (RainDance Technologies) following the manufacturer's instructions. In brief, primer panel droplets (MethylSeq Solution, RainDance Technologies) were dispensed to a microfluidic chip. The DNA template mix was converted into droplets within the microfluidic chip. The primer pair droplets and template droplets were then paired together in a 1:1 ratio. Paired droplets passed through an electric field inducing the discrete droplets to coalesce into a single PCR droplet (26 pl); approximately 1 million PCR droplets are collected per sample.

PCR droplets were processed in a PTC-225 thermocycler (MJ Research) as follows: 94° C. for 2 min; 55 cycles of 94° C. for 30 s, 54° C. for 45 s, 68° C. for 80 s; followed by 68° C. for 10 min; 4° C. until further processing. The ramp rate was set to 1° C. per second. Following PCR amplification, 70 μl of droplet destabilizer (RainDance Technologies) were added to each sample to break the PCR droplet emulsion and release the amplicons contained within the droplets. The solution was mixed well and incubated for 15 min at RT. Samples were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) following the manufacturer's protocol. For each sample, 234 μl of beads were used. Samples were eluted from magnetic beads in 40 μl of Buffer. The integrity and concentration (fragment range: 120-300 bp) of purified amplicon DNA were assessed using a High Sensitivity DNA Kit (Agilent Technologies) on a 2100 Bioanalyzer (Agilent Technologies).

Methylation Array

500 ng of DNA was bisulphite converted and hybridised to the Infinium 450K Human Methylation array and processed in accordance with the manufacturer's recommendations. DNA bisulphite conversion was carried out using the EZ DNA Methylation kit (Zymo Research) as per the manufacturer's instructions. Samples were processed in a single batch. R statistical software (version 2.14.0 [35]) was used for the subsequent data analysis. The ChAMP pipeline was used to extract and analyze data from iDat files, samples were normalised using BMIQ [36, 37]. Raw β values (methylation value) were subjected to a stringent quality control analysis as follows: samples showing reduced coverage were removed and only probes with detection levels above background across all samples were retained (detection P<0.01). DMRs (Differentially Methylated Regions) were determined using Lasso [38, 39].

High-Throughput DNA Sequencing

The pooled sequencing library (12 pM) and custom sequencing primers (0.5 μM) were applied to a MiSeq-cycle PE consumable cartridge (Illumina) according to the manufacturer's protocol. The DNA sequences of the custom sequencing primers are provided in Table 2 below. Sequencing was performed on a MiSeq DNA sequencer (Illumina) using 75-bp paired-end reads.

The RainDance ThunderStorm®. System was also used for the sequencing of nucleic acids (see the RainDance ThunderStorm System website on the world wide web).

Data and Statistical Analyses

Sequencing adapters were trimmed from the raw sequencing reads using the fastq-mcf tool of ea-utils v1.1.2-537 [60]. Trimmed sequencing data were mapped to an in silico bisulfite-converted human reference genome (GRCh37) using Bismark v0.7.12 [40, 61]. Methylation information was extracted using the methylation_extractor tool of Bismark v0.7.12 [61]. Targeted DNA sequencing analyses were performed using the R package TEQC v3.2.0.25.

Example 1: Methylation Profiling of Low and High Trade Bladder Cancer

The epigenetic alterations associated with bladder cancer were initially defined by performing genome-wide DNA methylation profiling on DNA from 81 high-grade and 30 normal urothelium.

Supervised analysis, using a Wilcoxon rank-sum test to assign directionality, was used to identify MVPs (Methylation Variable Positions) between bladder cancer and normal tissue. MVPs were selected on the basis of statistical significance (Wilcoxon P-value>0.001). An additional filter of Δβ>0.30(+/−) was applied to compensate for not taking into account the absolute difference in methylation between the groups. The cut-off is empirically defined to result in a false discovery rate (FDR) of <2% and reduced candidate loci to those with largest methylation differences and therefore greatest potential for a discriminatory effect. A total of 9786 MVPs met these requirements (1746 hypermethylated MVPs, 8040 hypomethylated MVPs) (FIG. 1).

Example 2: Bladder Cancer Specific Urinary Biomarkers

To define a DNA methylation biomarker panel, those loci which were determined to be methylated (β>50%) in at least 50% of cancers and unmethylated in normal urothelium (β<10%) were identified. By “methylated (β>50%)”, as discussed herein in relation to the development of this initial DNA methylation biomarker panel, it is meant that for any given locus, >50% of cells in a patient sample are determined to be methylated with respect to that MVP. In order to remove potential false positive biomarkers and better define alterations which are bladder cancer specific, whole blood and urine from 10 patients attending hematuria clinics and who had a confirmed non-cancer diagnosis was also profiled. Subsequently, any loci which showed any methylation (β>10%) in DNA from these non-cancer control urine and bloods were removed. A maximum of 432 loci were identified which are unmethylated in non-cancers and methylated (β>50%) in the majority of cancer tissue.

To derive a bladder cancer DNA methylation signature for detection we used the Random Forest framework, which resulted in a classification signature consisting of 150 CpG loci (FIG. 2, see Table 1 below). Using this core set of 150 markers we performed an internal cross validation of the classifier with predicted likelihood values i.e. likelihood of a sample being cancer or not for each sample independent of its relationship to the group of samples. This resulted in a cross validated sensitivity of 100% and specificity of 100% for the detection of cancer, showing that 150 epigenetic loci can clearly stratify normal urothelium from bladder cancer (FIG. 3, FIG. 4).

To determine sensitivity of the 150 CpG (MVP) marker panel for detection of bladder cancer using the classification algorithm, we assessed the methylation profiles of a further 179 bladder cancers (144 muscle invasive and 35 non-muscle invasive) and 20 normal cases. The panel correctly classified all bladder cancers, with a resulting sensitivity and specificity of 1.

Example 3: Validation of Detection Panel

To test the 150 loci panel (epi-signature) for the detection of bladder cancer in urine samples, DNA from urinary sediment cells was obtained from a cohort (n=86) including 52 patients with bladder cancer (low-grade=27, intermediate/high-grade=25) and 34 non-cancer patient controls. Applying the epi-signature to this validation cohort of give a sensitivity of 95% and specificity of 96% and an AUC of 97% for the detection of bladder cancer in this independent validation cohort (FIG. 5).

The large marker panel was also compared to the best performing single markers from the training cohort, this includes CpG loci from regions previously published as potential urinary biomarkers in genes including OTX1, COD1 and MEIS1. For each CpG a methylation threshold was defined, based on the highest β-value obtained from non-bladder cancer controls in the training cohort. This value was then used to predict the likely presence of bladder cancer in the validation cohort. The best performing single markers were combined and a predictive classifier developed to explore the potential for an “oligo panel” based on 3, 5 or 10 markers. Although sensitivities improve over single markers (best single marker 72%, best combined marker 70%), they still do not reach the required level to replace cystoscopy.

These data show that although single markers perform reasonably well alone or in combination (FIGS. 4-6, Table 1), the sensitivity to detect cancer using single loci or an oligo panel approach is limited and below a detection level desirable for clinical utility. Use of a large panel of markers out performs that of a single marker panel and Table 3 shows the sensitivity, specificity, AUC, PPV and NPV for the top 10 best performing markers and the combined 150 loci signature.

Example 4: Validation of Detection Panel Using High Throughput Technology

RainDrop BS-seq [31], allows large scale targeted bisulphite sequencing of a large number of regions (up to 20,00 unique amplicons) in parallel. This technology has been validated previously and shown to be highly correlated with the 450K methylation array, and its utility with low template input has also been validated [31, 32]. A bisulphite converted sequencing primer panel was designed to measure the methylation state of the 150 selected genomic loci (see Table 2 below). Primers were designed to interrogate both Watson and Crick strands independently where possible. Validation of the urinary epi-signature was conducted using RainDrop BS-seq in a second independent cohort of 96 cases. DNA from urinary sediment cells was obtained from 26 patients with bladder cancer and 64 non-cancer patient controls. Methylation score for each of the 150 loci was generated using the Bismark algorithm, using this data the urinary epi-signature predicted the likely presence of bladder cancer with a sensitivity of 96%, specificity of 97% and an AUC of 0.96.

Combining the methylation data from all validation samples allows an increase in the number of samples tested. Combining samples allowed assessment of 176 unique urine samples, 98 non-cancer urines and 78 cancer urines. The urinary epi-signature predicted the presence of bladder cancer with an AUC=0.98, independent of profiling technology (FIG. 7).

Conclusion

Biomarker-driven early non-invasive detection of bladder cancer has the potential to radically improve the management of this disease. Highly sensitive and specific assays have the potential utility in both the detection of de novo disease in patients attending haematuria clinic and also in the screening for recurrence in existing bladder cancer populations.

Several non-invasive tests are commercially available, and are based on cytology, FISH 311 analysis, and detection of mutations. Despite being FDA approved, the tests have reported sensitivities of 54% to 86% and specificities of 61% to 90% [12, 13, 41]. Performance characteristics are not sufficient to replace cystoscopy and therefore have not been taken up into clinical practice. There is therefore significant room for improvement and development of novel biomarkers, combinations of biomarker panels and the use of novel technologies may be most helpful for this purpose.

DNA methylation patterns are highly cell-specific and the ontogenic stability of these epigenetic events make DNA methylation an ideal biomarker for the detection and diagnosis of disease. Changes in global DNA methylation patterns are a common feature of neoplastic transformation and is a frequent event in bladder cancer. Previous studies have shown that methylation changes between bladder cancer, both non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) and normal urothelium are reflected in urinary sediment cells from bladder cancer patients, and as such could be a useful diagnostic marker. Several studies now have shown the utility of urinary epigenetic markers in the diagnosis of bladder cancer. However, although these studies have shown good sensitivities and specificities, they have failed to be taken up in clinical practice, predominately because they still do not command the performance characteristics to replace cystoscopy. DNA methylation biomarker assays (along with other DNA based markers, e.g. mutations) have been limited by the low resolution of primary analysis in identifying putative biomarkers, using either a candidate approach or a low-resolution microarray based platforms, and also by the limitations in the technology available for analysing candidate markers in urine. This has resulted in single/small biomarker panels being interrogated in the final biomarker panel [15-23]. However, in order for small biomarker panels to show the sensitivity and specificity to match those of cystoscopy they rely heavily on a low intra and inter tumour heterogeneity across a wide spectrum of disease states [42].

Novel technologies, such as next generation bisulphite sequencing and large scale multiplex PCR, now allow for larger panels of epigenetic biomarkers to be utilised [31, 32]. In order to define the epigenetic alterations involved in bladder cancer development and define a biomarker panel one of the largest unbiased genome wide DNA methylation screens of bladder cancer to date was carried out. Besides allowing an insight into the epigenetic alterations driving bladder cancer development, from these data a panel of the epigenetic biomarkers which has high sensitivity and specificity for detection of bladder cancer was also identified.

A biomarker panel of 150 single CpG loci was defined, which are predictive of bladder cancer. Although only a relatively small cohort, these data show the utility of a using a large panel of epigenetic markers, compared to single marker and small panel biomarker panels. With a sensitivity of 96% and a specificity of 97%, the negative predictive value (NPV) of this test is 97%. This is 1.2 to 8.7 times superior to what can be achieved by PSA (prostate-specific antigen) testing (PPV=30%-43%), mammography (PPV=9%-19%), or fecal occult blood screening (PPV=6%-11%; [43-48]) and similarly to that of cystoscopy (PPV=66.7%-98%) [49].

The inventors have shown that by applying a large scale highly multiplexed next generation assay, which is both highly sensitive and quantitative, the presence of bladder cancer in urine can be detected with a higher sensitivity and specificity than previously published methylation assays, and has a PPV comparable to cystoscopy.

The invention demonstrates that the combination of novel technologies, which allow the interrogation of larger panels, and bladder cancer-specific epigenetic biomarkers can be utilized to detect bladder cancer, allowing a reduction in the number of cystoscopies and consequently improve the quality of life for the patients as well as decrease health care expenditure. Furthermore, the utility of large panel assays allows for the potential of multiple clinical parameters to be evaluated from within the same data. For example, the stratification of tumour grade, recurrence or progression of non-muscular invasive disease, the likely response to therapy for muscular invasive disease or the differential diagnosis of multiple conditions.

Example 5: Optimisation of DNA Sample Handling and Processing

Optimisation studies were performed in order to maximise DNA yield from samples of patient urine. DNA was incubated with proteinase K for either 1 hr at 56° C. or for 48 hours at 21° C. Incubation was performed in the presence of RNAse A, 100 mg/mL. An increase in both amount and purity of the DNA was observed with the extended incubation protocol (FIG. 8D).

The UCL home urine collection kit was compared with a commercially available kit (Norgen, Cat #18124, see the Norgen Biotek Corp. website on the world wide web). The standard UCL urine collection tubes contain 70 mg/ml of Stabilur.TM. urinary preservative. This study was performed on urine from healthy volunteers; half of each sample was treated with the UCL standard method and the other half with Norgen preservative.

Little difference was observed in integrity of DNA from urine preserved with the two methods. No difference in DNA purity or yield was observed between first void samples and samples voided at other times (for concentration vs. time c¹ ₍₁₎=0.255, p=0.614 and for purity vs. time c¹ ₍₁₎=1.046, p=0.306).

However, an apparent increase was noted in DNA yield over 8 days in urine treated with Norgen preservative, whereas DNA from urine treated with the UCL standard protocol was of similar amount irrespective of time.

An apparent increase in urine DNA yield with the Norgen system was found to be attributable to bacterial growth over time whereas growth was effectively inhibited using the UCL established protocol (FIG. 8E).

Example 6: Additional Validation of Detection Panel Using High Throughput Technology

96 urine samples from 32 confirmed bladder cancer cases and 64 non-cancer cases (Validation Cohort 2) were analysed as described above. Sequencing of the 150 UroMark loci described above was performed following bisulphite conversion using the RainDance ThunderStorm® System. Statistical analysis was performed as described above, and the resulting ROC plot is shown in FIG. 9 (AUC=0.96, Sensitivity=0.97, Specificity=0.97, NVP=0.98).

In a yet further study, 92 urine samples from a cohort of haematuria and known cancer samples (Validation Cohort 2) were analysed as described above. This cohort consisted of 27 confirmed cancer cases and 65 non-cancer cases. Again, sequencing of the 150 UroMark loci described above was performed following bisulphite conversion using the RainDance ThunderStorm® System. Statistical analysis was performed as described above, and the resulting ROC plot is shown in FIG. 10 (AUC=0.955, Sensitivity=0.98, Specificity=0.97, NPV=0.97).

TABLE 1 Table 1 provides a list of 150 MVPs (CpGs) as used in the methods described herein. Provided for each CpGis the Illumina Identifier (Ilmn ID),  the chromosome number (CHR) and chromosome position (MAPINFO) specifying the location of the CpGin the human genome,  the gene name (if available),  a forward sequence encompassing each CpGand a corresponding SEQ ID number. The cytosine of the CG dinucleotide motif subject to modification is identified in bold and in square brackets in each sequence. The CpGs are listed in rank order from 1 to 150. The rank order is in respect to the number of tumours in which a given locus is methylated. Thus,  the MVP corresponding to SEQ ID NO: 1 is methylated in the largest number of tumours,  whilst the MVP corresponding to SEQ ID NO: 150 is methylated in the lowest number of tumours. MVP UCSC_REFG SEQ ID No. Ilmn ID CHR MAPINFO ENE_NAME FORWARD SEQUENCE NO. 1 cg25622366 2 63281139 OTX1 TTGCGAAGGCCGAGATCTGGGCCTGCCAGGGGCCTGCCCGAGTCCTCTATCGCGGGTCCA[CG] 1 TGGCCACCAATGACCCGCGGCGCCCCCGCGTGTCCCCGCAGCCACTCCGCGGAAGCAGCG 2 cg20302133 1 111217194 KCNA3 CCTGCTCGCCCGEGGCCGGCAGTGAGGGCGGCAGCGGCTCGTAGCGGTCGCAGCCGCCGC[CG] 2 CCACAGCCGCCTTGAGGCGGGGCCCCTCCACCATCGGCCACCTCCGGCTCCAGCAGGTGG 3 cg13046832 14 29254680 C14orf23 CCGGGACCCCGTCCTTCTTTCCCCTTCAGTCTTCAGGGAGGGGGAGGCGCTCCGCATTAG[CG] 3 GGGCAGTTCAGCAACCCCGACCCCACCCGCGTGGCTCCAGGCCCAGGGGTCCGTTCACTT 4 cg17945976 2 66667433 MEIS1 GTGGAGAGCTCACTCTGCAGAATAAAATCAAGAACACCACGGTTGTAGCTGCTGTGGACT[CG] 4 GCGGTCTGAGCAGCCTCCCCGAGAGCCGTAGTTGCTAGTAGAAGTAAGATTGAGCTCTGG 5 cg21472506 2 63283967 OTX1 CTCCAAATCAAAACCACTAAGAGTTCCTCCCGCGCAGACTGCTGCCCCTTCAGCTGCCCT[CG] 5 ATTTTGCTCCACGCCTGCCGGCCAGAGCCTCCCGGCGTTTCTTCCGCCCCAGCGGAGTGC 6 cg26492446 20 61638574 BHLHE23 GGAGCGCTAGTACTTGCTTCTCGACTCCCCGGCCGCCGCCTCCGGCCGCCCCGGGGATTC[CG] 6 CCACCAAACGCACGCGTCCCAGGTGGGCACCCGCCTCGGTCCGTCTTTGAGTCTGACCCT 7 cg11142705 3 42306974 CCK GCCTCTCTTGACGCAGCTGTAAAATGCGGATGACACCATCTGGTTTTGCTCAGAGGAATC[CG] 7 GTTTGGGAAAGGGATGTGTTTTCTTCCCGGGCCAAGTTACCACCACCCGCGGCGCCCACT 8 cg26970841 16 85932666 IRF8 TGGCTGCCGGGGGCGGGAAAGTGATTTCTCGGAAAGCAGAGCACTTCGAAGAAGGCCGGC[CG] 8 CGCGAGCCAAGCTGACGCTATTGGTCGGTGTGGCCGTCGCTCTGCGCACCGCCCGTCCCC 9 cg23497016 2 66666684 MEIS1 GCAGTGGAGGGGACGAGGGCTTGTCGGGTGGGAAACTTAATTCAAAATGGCTGCTGGAAA[CG] 9 CTTGGGTTTTATTCGTAGCAAATGTTGCCAATTTCTCCGGCCAGATACGCTAAACCGATC 10 cg19178853 4 174450408 NBLA00301; GGCGGCGGCGGCAGCTGCGGCGGCGGCGGCGGCAAACGGGTAGCCCTCGTGGTGCACCAC[CG] 10 HAND2 GGTGGTGGGGAAAACCACCTACCAGACTCATTTCGCCCTCCGCGCCCCTCCACGCGCCCC 11 cg14428146 8 23563925 NKX2-6 TCTCCAAGACTGCCTCACAGGGACCCCCAGGAGGCTCCGAACCATCCAGCTTTCTGTCAC[CG] 11 CCGCCGCCACCAGCGTTGTGAACCTCTGACCCTCGCGGCTCTGCGTCCATTCTCAGGTAC 12 cg16887264 4 147561775 POU4F2 TCCCACCGCGAGAAGCTCACCAAGCCTGAACTCTTCAATGGCGCGGAGAAGAAGCGCAAG[CG] 12 CACGTCCATCGCTGCGCCAGAGAAGCGCTCGCTCGAAGCCTACTTTGCCATTCAGCCTCG 13 cg06392169 6 391936 IRF4 CCAGAGGTTCGACCTCCAGGGCAGCGCAGGGTACCCCGGCTTCGGAGCGGGAAGGGAGCG[CG] 13 CCCCGTCCTGGAGCTCCGACTCCCACCCCATCTGCGCTGAGCCGGAGGCGCTGGTTTGGG 14 cg11433622 2 66667549 MEIS1 CTCTGGGCTTGTTTTCAGCTTATTTAATTCACACTGAAATGTTTCTGCGCGGGACGAACT[CG] 14 GTGTCACCGGGTCCCTCCCGGAGGGTTACTTCCTGCCCCCGACAGTGTAATGAGGCAAGA 15 cg01791874 5 16180055 Mar-11 CGGGGGTGACGGGGCGCGGGCGCGGGGTGGGCTGGGGGCGCGGATCAGTGGGACGGAGTT[CG] 15 GGGTTCGGCTCCGAGCGGGCGGGCTGGAAGTGGGGGATCCCTCAGCCGCCTCCACGGGCC 16 cg23180938 5 115152485 CDO1 GGAGGACGAGGCGGAGAGCCACCCAAGAAAGGTGGCGGAGCCCGGGAGACCCTGCGCGCA[CG] 16 GCTCACCCGCACATCCCCGGCTTCCCCGGGCTCCGCGCCTTCCCAAGAGCCCCGTTGTCT 17 cg26990102 2 119606746 EN1 ACTTGTCTCCGGATTCGTTTTGGAGGACCAGTATCCGACTATGCTGAAGCCTGCGGTGGC[CG] 17 AGAAACGCCTCAGCAAACGACGCCTTCGGGGGTGTCATCTTACAGCTCCAGCGGACCTCC 18 cg11595545 1 111217497 KCNA3; GGGGAAGAGGCGGCAGCGGTGAGGCCAGGTCGCTCCTCCTCGCGCTCCCCGCCCTTTCGC[CG] 18 KCNA3 CCTCCGCCCCCGAGCCGAGCCCACCGCCTGTTGCAGCCAAAGCCGCGATGCTCTGTCTGG 19 cg00986824 12 75601465 KCNC2; KCNC2; GTGCGGTAGTAATTGACCACATAGGCGAAGACGCCCGGGTGCCGGTCGAAGAAGAACTCG[CG] 19 KCNC2 GCCGCCACCGGGATCGTCGCTGGCCCTGCCGCCGCGGGAACTGCAGTTGCCCGCGCCGCC 20 cg09462924 2 66666470 MEIS1 GGCTCTCCGAGGGCCTTGGGGTTGGGATCCCTAGGTGCAGCCCGTTGACAGTCGGCCCCA[CG] 20 GCCATGGACGTCCTTTCCCCAAGTTAGCTGAGCGCCTGCCACCGAGATCCCCCGAGCCTG 21 cg07790615 1 237205174 RYR2 AGATTAGCCACCCCGTGCGCCCAGGTGAAAGATATCATTCTTCCGTGCGATCCGAAGTGC[CG] 21 TGGAAGTTAGTGCCCTAGCCCAGTCCAGGAGGAAGGGCCGTCGTGCCGGCGGTTTTAAGC 22 cg06785999 14 60975964 SIX6; SIX6 GCCGACCCCGAACCCCAAGCCGCGGAGCCAGCACCTCCTCCAGTCGGGGTCGTCCGCTCC[CG] 22 GCCGTTGAGCCACCGCCGCCACCCGGTAGTGTGTCCCGCTGCCCCAATCCGCCTCATCAA 23 cg04550737 1 119530600 TBX15 GAGACTGCGGCTCGCGGGTCTCTCCACCCTCCCCCCGCGTCCTCCTCCGCCCTCCTCTGC[CG] 23 GATCCGACCTGCGCCCCTACGCTGCCCCAGCTGCTAGGAACTAGCGCCCCGAGCGCCGCC 24 cg19978181 5 76923988 TTACTTGGGAGGTCCCGGGTATCTGAAGCGGATCCCGGGTCTGGGGACATGAAGGGGCGC[CG] 24 TGGCCTTAGGGAAGGCCCCAAAGAGGCCTAGGCCCCGGAGGAGGCAAGAGCCGCGGCCTA 25 cg23065934 5 16180266 Mar-11 GGGGAGACGCCGCGGCTCCAGAAACCGTTACTGGATCGGCCGGTGGGATGTGGCGCGGGC[CG] 25 GGTGGGGCGCGACAGTCTGAGCCGAGACCCGCGTGGGCTTAAGGGTGCGCGAGGCGGGTG 26 cg14470895 5 115152431 CDO1 CGGGCCCCTTTTAAGCGCTTGGAGTCACTAGGAATGTACCAACGGCCCTCGGAGGGAGGA[CG] 26 AGGCGGAGAGCCACCCAAGAAAGGTGGCGGAGGCGGGGAGACCCTGCGGGCACGGCTCAC 27 cg11036833 5 115152494 CDO1 GGCGGAGAGCCACCCAAGAAAGGTGGCGGAGGCGGGGAGACCCTGCGGGCACGGCTCACG[CG] 27 CACATCCCCGGCTTCCCCGGGCTCCGCGCCTTCCCAAGAGCCCCGTTGTCTCCGGCGTCC 28 cg23727983 11 125774082 DDX25; ACACGGGGCGTAAAGCGCGGCGGGGAGTCCGGGGGGCTCCCGCCTGGAGGGCTGTGTGAG[CG] 28 PUS3 GCGGGCCGCGGGGCGGCGCGGGGGGCGCTCTCCACTCTGCGGAAGCTGCCCCCTCTGCCC 29 cg16732616 1 50886782 DMRTA2 CGGCGCACACTGAACCGAAGACCTCGTAGGCGGGCCTCGGGGGGATGATGCCGTTGCCGG[CG] 29 GCCAGCGCCAGCCCCTCCGCAGTGCCGTAGAGCAGCTCCAGCTCGCGCGCCTCGTTCTCC 30 cg16422098 10 15761881 ITGA8 CCCTGCGGGGCAAGGGGGGCTGGTGGAATCTGGCGGTCCCCAGCTCCCCGTGTCCCGGGT[CG] 30 GTCCGCTCGGCGCACCCGTGGTCACAGTGCCCGGCGTCTGCTCCCACCCGCCCGCCCGCC 31 cg13620034 8 72755052 MSC AAAAAGAACGTGAGATATTAGAGAGAAACGATTGTCTCAAACCGAAACAGCTCTCCTACG[CG] 31 AACCCCAGATATTCCTGACTTGGAGTAGCTAAGATTTTATCAGCATTCTGGGAATTTGCA 32 cg09424526 2 223163809 PAX3; PAX3; CTTTTACCCAAAGCTTGGTCAGGAGCCCTGAGCTGCGATTGGCCGACGGGTAGACCGTCC[CG] 32 PAX3; CCDC140; GGTGGCGGAGACACGCGCTGATTGGGCAACAGCGACCACTTTCTCTTCCCATCTCTGGTG PAX3; PAX3; PAX3; PAX3; PAX3 33 cg09813525 13 53420386 PCDH8; GGCGTGTCCCATTGCAGCACCGACCCGGACACCCCGAGCCGAGAGCCAGGCGGGCGGCAA[CG] 33 PCDH8 CTCCGGGCTTCCTGCACTGGCAGGCGCAGCCGGCCCACGCCCGCCCCCTGCTGTTACCAC 34 cg18082337 3 147113726 ZIC4; ZIC4; GATTTTCTGATCTAGCAAAGACCTTCCCACACCCCGGGAAAGGACAAGGGAAGGGCTTCT[CG] 34 ZIC4 CCCGTGTGCACGCGGATGTGATTTACAAGTTTGTATTTGGCTTTGAAGGGCTTTCCCTGG 35 cg27655158 4 1396593 GTGAAGTTCCGCAAAGTGCTTCGCAGCGGGCCGAGCCCGCGGGAGCCACCTGCCCGGCCC[CG] 35 ACGCGCATGGTCATTTATAAATTTAAAACTCTTCCGTAGCAACCGGTTATGTACAGAGTC 36 cg26802289 7 20817859 GTGGCGCTCGCTGTCCGTGCAGCGGGCGGAGGCGGCCGCGGTGCCTTTGTGTGCGGTGGG[CG] 36 CGGCGATGGGCTGCTCCGGCCCGCAGCCCGAGGGGAGCGGCGGGCCCGGGCCGGGGCCAG 37 cg18074954 18 44787492 CCTGTCCGCGCCGCCCGCGCGGTCTCCGGCCCCCTDCGCGAGGTCCTCCCTCCTGCCTCG[CG] 37 CTCCACGGCTCCTCCGCGCTCTGCCTCCCGCGGCGCCTGCCGCCTCCCCTGCGCGGATCA 38 cg25947619 15 86233220 AKAP13; TTACGATTTCACTAATCGCAGCTCAAAGCTGCTGAGCTCTGCCCTGACGGCATGCGCCCC[CG] 38 AKAP13; CTTCTGTGAGAGCACGCTTCCTGGCATGGGGGTCGAATCATGTCCATAAAATGGGCTCGG AKAP13 39 cg15384598 14 24045549 JPH4; JPH4 TGCCTCCCTCGTCGCCCGGCAAGGGCAGGGGCGGGGGTGGCGTCGGGGGGTCGCTGTGGC[CG] 39 GAATCCAGGGAGGTGCGGCGGGGCGAGCGCAGCAGCGCCGCCTGATGGTAGGGCACACTC 40 cg16178603 2 66667101 MEIS1 CGAGCCGGGGGTGGCGGGCGGGGACGTCTGCTCGTCAGAGTCATTCAATGAAGATATAGC[CG] 40 TGTTCGCCAAACAGGTCAGCAAAATAGATGTTAAAAAGTAAAAGAAACAAAAAGAGAGCC 41 cg23241781 14 70653964 SLC8A3; SLC8A3; TTTAATTCTTCTGCCCGGGAGACCTAACCGCCGAATGCGTTCGGAGGTATGTTTTTTAAT[CG] 41 SLC8A3; CCAGGAAAGGGGGAGAGAGAGAGAAAGAGAGAGAGAGAGAGAGAGGAAAGGAGGAGAGAA SLC8A3 42 cg01419831 2 162283705 AGGAGGAGCACGTGCGGTCGGTGGGGCGCAGGGCCGGGGAGCCAGGGGGGTGCCGGGGCT[CG] 42 AGGCCGCGGGGCCGCGGGGGCCGCGCTCTGCTCTCCGCCCAGGCTGGGCCACTGGAGCGC 43 cg17039236 18 77548049 AGGCGAAGGCCGCCCCGGGAGAGCGGGGTCCCGGGAGAGCGGGGTCCCGGCTGTGGGGGA[CG] 43 CGGGCCGAGGCTGTCGCGAAGCCGCTGACGGCCGAGGCGCTCCCGGTTTTCGCGGCGCAC 44 cg21901718 5 16180076 Mar-11 GCGGGGTGGGCTGGGGGCGCGGATCAGTGGGACGGAGTTCGGGGTTCGGCTCCGAGCGGG[CG] 44 GGCTGGAAGTGGGGGATCCCTCAGCCGCCTCCACGGGCCGGCCCCGCGCTCACGTCGGTT 45 cg25191628 1 50886949 DMRTA2 CGCTGGCGCTCCGCGATGAGCGTGCACTTGGCGCACAGGCAGTCCTTCCAGCGACAGTAG[CG] 45 TTTGTGGCCCTTGAGGGCCGACACCACGCCATGGTTGCGACAGCGCGCGCACTTGGGGGT 46 cg27277463 12 62585031 FAM19A2 CCGCGGTCCTGCAGTTGCCGCTCCGGTCCCCAGCGCTGGCCGGCGACCCGAGGCGCGGCT[CG] 46 CACCTACCTGCAGCCCCGCTTCCCGGTGGCGGCAACACCTAGCGATGCTCCTGCAGCTTT 47 cg05311410 7 27225523 HOXA11AS; CTGGTGGCTTGTCCGATTTGCACGGTGACTTGATTACACTCTCTCATTCATGGTCACTTC[CG] 47 HOXA11 AAGCGCTTTAGTGCCTTCCGTCCCTAAACCGCCAACAGCCAGAACGGCTTCTCCCCGCGG 48 cg23229261 2 63284066 OTX1 TTTCTTCCGCCCCAGCGGAGTGCGCTGGGGCGCGCCAGGGCTAGGCCCGCCGGAGGAGCG[CG] 48 TCCCCAGCCTTCCGCGCACAGAGCCGCATCCCGCCCCGCCCTGCGCTGGACTGGTTCAAG 49 cg10397440 8 57359258 PENK; PENK; CCCGGGGCTTAACGGCTGCTGGAGCCACTTTATAATTAGCCCCAAACCGAAGGAGGCGCG[CG] 49 PENK CGCCCCAATCGCCGGCGGGCTGCAGCTGACGCAGGCCCTACGCCAGCCCCGCGCCGACGC 50 c018326021 10 106401479 SORCS3 GGCGAGCGGCGGGGCCGGGGCATCCCAGCTCCTGCCAAGCTTGGCGGCGCGAGGAGGAGT[CG] 50 CCGGGCGCAGCCCCCAATCACCCAGGAACGCGGGGACGCCTGGGCCACTGCTCCGGCCGA 51 cg06533244 6 27258460 CACAGGTTTCCGTGGTGTAGTGGTTATCACATTCGCCTTACACGCGAAAGGTCCTCGGGT[CG] 51 AAACCGAGCGGAAACAACTTGCAATTTTTCGGGGTGTTTCTGTTTTCCAAGATTCCCTTA 52 c003078363 12 54408664 GTAAAAACCCGTTTTATGGGGGAACGTAATTGTGAGCGGGATGCGCTCTCTTTAGAATCG[CG] 52 TCCTCCCAAATGCTCCCGCCGTCCCATTACCGGAATGGGGACCATTCGGCTGCTGCAGAT 53 c001796166 14 95239586 GGCCCCGCATCGCCTGGCGCAATTGGAATAACAAATGCGACGCACGCAAATTGTCCACCT[CG] 53 TGTTGCTAAGCGATTGTTTGTCGGCCCCGCACGCACAGCTCAGCATGGGAGGACCGCGAG 54 cg00339556 5 16180048 Mar-11 GGCGCGGCGGGGGTGACGGGGCGCGGGCGCGGGGTGGGCTGGGGGCGCGGATCAGTGGGA[CG] 54 GAGTTCGGGGTTCGGCTCCGAGCGGGCGGGCTGGAAGTGGGGGATCCCTCAGCCGCCTCC 55 cg03978375 16 85932668 IRF8 GCTGCCGGGGGCGGGAAAGTGATTTCTCGGAAAGCAGAGCACTTCGAAGAAGGCGGGCCG[CG] 55 CGAGCCAAGCTGACGCTATTGGTCGGTGTGGCCGTCGCTCTGCGCACCGCCCGTCCCCCC 56 cg26013553 1 111217406 KCNA3 CTGCTCGCTGGGCGCTGAGGAGGGTGGGCGCGGTGGCGGGCTGAGGGCGGCGGCGGCGAG[CG] 56 CAGAAGGCTGAGGCGCTCGTCCATGCGGCGGGGAAGAGGCGGCAGCGGTGAGGCCAGGTC 57 cg09734791 8 72756155 MSC ACTGCTTGCACTCTGCGGCTGAGCCCTTGGCCGGGAGGGGCTTCTTGCCACCACCGCCCG[CG] 57 CTACCACCTGCGCCGCCGCCCCCAGCCACACGGGGCCGCTTCCTCTTGCAGCCTTCCGCG 58 cg12040830 11 112833773 NCAM1; GGCGGGCCAGGGAGCACCCAGTGCGCCCCCTCCGCGGGCGGCACAAGAGCAGCGCTCGGC[CG] 58 NCAM1; CGCCTCCAGCCAACTCGGGTCCCTCCCACGGCGACCAATCAGTGCGAAGCTGGCTGGGC NCAM1 59 cg18617005 5 140787504 PCDHGA4; ATTTCGGAGACCGAATTCAAAATGAAAAACCGGGCTGCTGTCCCGCACGGAGCCTCTGGG[CG] 59 PCDHGA6; CCGCTGTCGGCCAGTGCAGAGCAAGCGCTGACGCCGGGGATCCGTCAGCCTCTGGCCTGG PCDHGA9; PCDHGA1; PCDHG58; PCDHGA5; PCDHGB1; PCDHGB6; PCDHGB4; PCDRGA3; PCDHGA2; PCDHGA7; PCDHG82; PCDHGB6; PCDHGB5; PCDHGB3 60 cg19597382 4 147559423 POU4F2 ACCTCCGCTCGTATTGGGCTGGGAGTTCAGAGCCGCGCGCAGAACCCGGGTTGGCCGCAA[CG] 60 TCTGTGTTCTCAGCGGTGGCCGGGAACCTGGGATCAGGGTCACCTGAGCTGACGGGGTGG 61 cg05134015 14 36973365 SFTA3 CCGGAGGTTCGATGGCCGCCGGGCCAGTGCGGGCTCAGAGGAAGACCCTGCAAAAAAGAG[CG] 61 CTCGCCCCCACCCCTGGAGCCGACCCTGCGCAGTAGGGCCGCAGCCGGTCCCCGCGGGCA 62 cg02700891 3 129693586 TRH, TRH GGCGCAGATATAAGCGGCGGCCCATCTGAAGAGGGCTCGGCAGGCGCCCGGGGTCCTCAG[CG] 62 CTGCAGACTCCTGACCTGCCGACTGCGGATCCCGAGTCCCCGGATCCCGGACCCATCCTG 63 cg13476854 1 119549263 GTCGGATCTCTAAATTATCTAATCTGGCGGCTGCGTACGACTCAGGGAAAGCCCTGGCCG[CG] 63 AGCTTTTTCACCAGGCTTGAGCTCAGCAGCCGGGCCCGCAGTGTTGCCGCCAGTGGGGAG 64 cg00017221 1 149719536 GTCTCTCCACCCGCTGCCGCCTAGCAAAGGCGCATCTTTAGGTCGGTAGTGAGGTGCCGC[CG] 64 GGACGCTGCAACTCGCTCCGGGACTTGTAAACCTGGCAGGTGTTCGAAGAGGGCCACTGG 65 cg05310764 6 27513479 CACTCAACCATTATAAGTTCACCCCAGCCGTCAGCGATGGCGTAGGTAGGTAGTCGTGGC[CG] 65 AGTGGTTAAGGCGATGGACTTGAAATCCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTG 66 cg04495995 7 19147157 GTGTAGAGAACAACAGTCGCTCCTTAGATATTACTCCAGGACGGAAACCTGATTGCAAAC[CG] 66 CTGTTCCTTCGAAACTTGCAAAACCCGGAACAGAAAACTCCCGCCCAGCCAATTTTAGCT 67 cg01642521 11 20618250 ATTCAGACCGAATGGCTGCGCGGTGATGGATGCGGATTTACGGCCTCCTTGGCTGCGGCG[CG] 67 CTGGGCCTGATTATCACTATAAACAGGCGTCCGCGGAGGGCGGGGGCGGAGGCCCGCGTC 68 cg24504927 12 85667353 TTAACCACAGAGTTGTTCTTGATTGTAAGGGACTTCGCCCACTTGGTTGAAGTGGAGAGC[CG] 68 GTCCTCATTCCAGACGTCCCGCACGGCAGTCGCTCATGGCTCCCTCCAGGCCGGGAGCCA 69 cg03276408 13 112712475 GCAGGGACCCGGCTGGCCGCCTCCCCTTGCAGGAAACAGGTGTTTGAACGCGATAGCGGC[CG] 69 CCAGTCAACTAAGGCATTAAAAGCTCGCTTTATAACATCGATTTCCTGGAGTGCGGTGGG 70 cg27501878 20 61638588 BHLHE23 TGCTTCTCGACTCCCCGGCCGCCGCCTCCGGCCGCCCCGGGGATTCCGCCACCAAACGCA[CG] 70 CGTCCCAGGTGGGCACCCGCCTCGGTCCGTCTTTGAGTCTGACCCTAGCGCAAGAGTCCC 71 cg07495363 2 198651076 BOLL; BOLL GGACTCCGAGCCGGGGCGTCTCAGGGGCAGAGCGCACGGCACAGCGGGGCGGGCGTGGGG[CG] 71 TGCGGAGCGAGGGCTCGGTTCTGGGACCCCTCTCGCCTTCTCTCAGACGGCTGGGGAGGA 72 cg06750832 1 111217691 KCNA3 CTGCGGGGCGCGCGCCCGCCTCCGCGTCCCCTTAGGATTCCCGCCCACCGCGCGGGCGCG[CG] 72 TCCCGCTCTCGGGGGCAGCCGCCGGGCCTGCATTTCTTGCAGCCCTCAAGGCCCCTCGGT 73 cg22474464 20 21492914 NKX2-2 GGAACCAGATCTTGACCTGCGTGGGCGTGAGGCGGATGAGGCTGGCCAGGTGTTCGCGCT[CG] 73 GGCGCCGACAGGTACCGCTGCTGCCGAAAGCGCCGCTCCAGCTCGTAGGTCTGCGCCTTG 74 cg20146541 1 248020697 TRIM58 ACTGCGGCCACAGCTTCTGCCTCAGGTGCATCTCCGAGTTCTGCGAGAAGTCGGACGGCG[CG] 74 CAGGGCGGCGTCTACGCCTGTCCGCAGTGCCGGGGCCCCTTCCGGCCCTCGGGCTTTCGC 75 cg02864844 7 149917263 GGATGCAGACGCACCAGCGGCTGCTCACACTCCCTCCACAAACCTGCCGGAGTCTCCACT[CG] 75 CCCGCCAACTGTAGCCTCCATCTGCGCCCCACGCCCCCGCACAAGCCCCCTCCGTCGCGG 76 cg12323723 X 136656581 GGCGCAAAAGGGGCCGCCCCCGTGCCGGGAACAGACTTTGAAGTGGGTTTTTAGCGCGCA[CG] 76 TGTGAGAGCCGGGCCAGGGCCGGAGCGGGGACCCGCTGGGAGGAAAGAGGAGGCTCCGGC 77 cg14996220 12 85673270 ALX1 CTCCCACCCATCGCCACCGCGTCCACACTCCGCCGCATTCCAGAGAAAAAGAAAAGGCCA[CG] 77 GCCTCGTAAAGCTCCCAGCTGGCCCGGACCCCGAGCTCCTCCCGGTTAGAAGCCGAAGCG 78 cg23151000 2 38302892 CYP1B1 GATTTCCTTTAAAGTACCTACCAGGCCACCCGCTACCTGTAATAATCCATCTGAAGAGGT[CG] 78 CCGGGGAGCGCCTCGGCAGACAGACTGACCTGCGGGGAGGTGCGGTTTCCAGTGGCGCGG 79 cg00963169 1 50513927 ELAVL4 CGGCGCAGGCCCCGCACCCCCGACTCTGCCCGCCCTCTGTTACGGACACCCGCTGGGCCA[CG] 79 TGGTCGCGACTGGCTTCTCCCAGCGGCCAGCCTGGCCACCCCGACTCCCAGGGAGGGGGA 80 cg13320291 10 118030970 GFRA1; GFRA1; TCGGCCCTCCGCCCCGGGGCATCCTGGCCTGAGCAACGACCCGGGCTCCCGGGCCACCCC[CG] 80 GFRA1 GCTCCAGCCACCCGCTCCGCCCGGCTGAAACTCAGGCGCTTTCCGAGGAGAAGTGCGGCG 81 cg13352750 7 27225123 HOXA11AS; TTCTCACCGAAAGCACGTAATCGCCGGTGTAACTCATGTTGGCTGGGGGGCCTCCCGGCG[CG] 81 HOXA11 CGCGGAGAGGCTGGGGTGCGCCCCCATGCAGCATGCTTGTGCTCAATTGCAGGGTCCTCG 82 cg26521404 7 27204981 HOXA9 GCTGAAGTCGGGGTGCTCGGCCAGCGTCGCCGCCTGCCGGGGAGGCTGGCCCAGGGTCCC[CG] 82 GCGCATAGCGGCCAACGCTCAGCTCATCCGCGGCGTCGGCGCCCAGCAGGAACGAGTCCA 83 cg07573209 15 76630095 ISL2 GGGTTGGGTTGGGGCTGGAGTAGCCGAGGCCGGCCTGGGTCCGGGCAGTCAGGCCTGACG[CG] 83 GCCCCGCGCCCTTCCCCGGCAGAGAAGCCCGGGACGGCCATGTGCGTGGGCTGCGGGAGT 84 cg10835584 18 55108852 ONECUT2 GCGCTTCCGGCGATCCGCCTGGGCGGCTGGGTCCGCGAAGCCAATGCGCTGAACGGTGCC[CG] 84 AGTCTTCCTAACTATCCTGTGCTTGGCCGTTGCCACTGGGCCCTGGTGACTAAGCCCAAG 85 cg09474331 19 54926805 TTYH1; TTYH1 TACCGGCCCTCAGCTTGGGTGCATCTCCTCCACCAGCTGCCCCGCGCCGACTTCCAGCTC[CG] 85 CCCGGTGCCCAGCGTTTTCGCGCCCCAAGAGCAGGAATACCAGCAGGTGGGACCGGGCGC 86 cg20870512 7 1272515 UNCX GTCCCAAATTCCCCGGGCCGCGGCTAATTATCGGGAGCTTGATGTTGATAAGTAAAGCGC[CG] 86 GAGTGCGGGCGAAGCATGTGTGGGGCTCCGGGTCCCTGTCTCCGCCGCCGCCGCCCGCGC 87 cg21678445 18 22930283 ZNF521 GAGGGGCGGTCAACTTCTGGGAATGGCCAAGAGGGGTGCTCTGAGGCCCGAGCCGGGGTC[CG] 87 GTGCCCGCGGCCGGCGGCCGGGGTCTGTTTACTCCGGCGACGTGCGGAGAGGACCCAAGC 88 cg05560435 5 172671526 CGCCGCCTTTCCCTCCTCGCCTCTTCCTTCCTTCCGGGTCGTGCCCTCCAACCTGCTGTG[CG] 88 TTACCGCAGCCAAGTTTCCACCGCCCGGCGGAGCGCATTGTGAACAGCAGCTGACAAATT 89 cg08382226 6 108440339 CCGACTCGGGATGACAATTGACGGGGATCAAGGGATTGCCCATTCTGTGCCTGTAAGAAC[CG] 89 ATTCGTGCCAGAGAAACTCATCAAGTGGAGGCGGAGAATAAAGACCGTTCGGGGGTAAAT 90 cg25510609 15 86233236 AKAP13; GGCAGCTCAAAGCTGCTGAGCTCTGCCCTGACGGCATGCGCCCCCGCTTCTGTGAGAGCA[CG] 90 AKAP13; CTTCCTGGCATGGGGGTCGAATCATGTCGATAAAATGGGCTCGGTTTCATGAAGTACCCC AKAP13 91 cg19650157 13 78493297 EDNRB; EDNRB; GAAAGCCGCTACTCCCTGGCTGGCTGAGCTACAGCTCCCGCAGCGCGCCCAGGAGTGCGC[CG] 91 EDNRB GAGATTCGGAAACCCGCAGAGACTTCTCAAGTCAGCAGGAACTTGGAAACCGCTGTTCCC 92 cg01283246 5 135266135 FBXL21; CTCGCTCGGGCGTGTTCCTGCGCCGACCGGACGGCCGGACTCCAGCACCTTGGCCCGGCC[CG] 92 FBXL21 CGAACGCTGAGCACGCGCGGAAACCCTTTAAAGGTAGCACATTTTTCGGGTGTCGCGGGG 93 cg20872937 18 74961968 GALR1 CTGGAAAAGCCGGGAGGGAGTCGGAGGCGCCAGCCCACTGGGGAGGTGGCGCTGGGCGCG[CG] 93 GGATGCGCGGGGAGCCTTCTCTGCAGGAGCCGCACAGTGCACTGCTGCGCGCTGGGCAGT 94 cg03698009 7 27204349 HOXA9 TCTCCCGTAGCCCTGCGGGCCGCTCTTCACTGCTCTCCAGACTTGGGGCCCTATCTGAGG[CG] 94 TCCCAAACACCAACTTCTGGCTCCTGGCCCCAACTCGAGAGGCTTCCAGCGAGGACGAAG 95 cg01423964 1 111217575 KCNA3; GAGCCCACCGCCTGTTGCAGCCAAAGCCGCGATGCTCTGTCTGGGTCTGGCGCGGTCAGG[CG] KCNA3 GGCTCCCGCACGGGGACGCCTCCTCCCTCCTTCTCGCGCTCTCCGCCCCCTCCCCTGCGG 95 96 cg19923650 5 172659730 NKX2-5; CGAAGTTGTTGTTGGCGGCGGCAGTGGCCGGCTGCGCTGGGGAAGGCCCGGCGGGGTAAG[CG] 96 NKX2-5; GCAGTGCAGCTGTAGCCAGGGCTGCAGGCCGCGCCGCCGTAACCCGGATAGGCGGGGTAG NKX2-5 97 cg13324546 8 23564031 NKX2-6 GTCCATTGTCAGGTACTGAAAGTTTTCGGGGCTGTTGGGCAGGCGCGGATGTGGCGAAGC[CG] 97 CGGGGCAGGTCCGGTCGGGCTGGAGTCGCAGGATGTCGTTGACCGAGAAGGGGGTGGAGG 98 cg27357571 21 34398226 OLIG2 CGCCCCCGCCCCCGCCACCGCTGCCGCCGTCGCCGCTGCCACCGGGCTATAAAAACCGGC[CG] 98 AGCCCCTAAAGGTGCGGATGCTTATTATAGATCGACGCGACACCAGCGCCCGGTGCCAGG 99 cg08448701 20 21686282 PAX1 CAGTGACGGGAACCAATGAGCTGCCAACTCGCGCGTCTCCGGCGTGACTGCCGAGATTGA[CG] 99 TGGAGGACACGTCAAATTGATTCCCGCACGCTGCAGCCTCCCGGTCAGACGAATTTCTCC 100 cg16002355 4 111544387 PITX2 ;PITX2; CTCCGCCTCCTCCCAGACCCTTCTCCGGGTGCGACTGACGTGGCTCCGCACCAATCAGGA[CG] 100 PITX2 CCCGAGCCGCGGTGGAGGGACTGTCCTGCCTGCACCTATCAGCAGTGCGGGGCCGGGCT 101 cg05302386 14 52734525 PTGDR; TTAGCACCCGGGCGCCGGGGCCCTCGCCCTTCCGCAGCCTTCACTCCAGCCCTCTGCTCC[CG] 101 PTGDR CACGCCATGAAGTCGCCGTTCTACCGCTGCCAGAACACCACCTCTGTGGAAAAAGGCAAC 102 cg17964510 2 175199694 SP9 AGGCGCAGCCAGCGGCACTTCAAAGCGGGTGCTCCTCGCACTTAGGCTGAGTTTAGCCGG[CG] 102 GGAGCCTGGAGTCCGCTCGGCACGAGCGCGGGGACGCGGGAGCCGCGCGGGACCCAAGCA 103 cg24031355 10 22634439 SPAG6; SPAG5; GGCCGCGTTCCGGTTCCGGTAGGTTGCCCGGGAGACGCGGGTACACAGAGAAGCGGCTCC[CG] 103 SPAG6; TCGGAGGCCGAGTCGTCGCCACGATCGCCCCCTTGGTGGACTCGCAGGCCGAGCGGCTTC SPAG6 104 cg24961583 3 27765409 GGGAGCGCATTTTCCGGCTGAGATGTCGGGACTCTGCTTCCCCAACCGAACGCGATCACA[CG] 104 GGAAACTCTTCGCCCACAACAGATGAGATGGCCAAAGGATTGCTGAGTGCGCACACGCAA 105 cg02167020 10 102899949 GGACTCATGCAGAAGAGGACATTCCGCAGGTAGGTACAATCCCAGCGCTGGGGCCTGGGG[CG] 105 TCCGGGGGGCGGCCTTTGAGCTTCCCGGATACCGCTCGCCTGCTCCCGGAGCTGTTCGGC 106 cg25026529 1 91183051 BARHL2 GGGGGAGTTAAAAAAATTTCTGAGAAAACTCGGAACTTGCGCTCCAGGAACGACTGCGCA[CG] 106 TGGCGCGGCGGTGGCGGCGCGGAGGACCCAGGCGAAGGCGAAGGCGAAGGCGAAGGCGCA 107 cg05783139 2 198650985 BOLL; BOLL GACTGCGTCGCCTCGGGTGGCAGGTGGCGGTGCGGGCGGGCGCTGCAAGCCGGAGAGGGG[CG] 107 CGGGAGGGCGAGTTTCGGCTGTGGCCCTGGGACTCCGAGCCGGGGCGTCTCAGGGGCAGA 108 cg25691167 7 19184961 FERD3L GAGGGCGTCTCGGGGAGGCCAGGGACAGGTCTGCGACGAAGTCCAGCACCGTAGTGTCCA[CG] 108 CAGCTCTCCGGATAGGCCGCCATCGCTTCGGCTTGGCCCTGCCTCTCATCGGTTTTCCGC 109 cg25951981 4 46995743 GABRA4 GTATACCGCTCCACACCCTTTCGTGCCCGCGCGCTGAAGGTTCTGGGGTTCGTATCCGCG[CG] 109 CTTGCGCTGCAAGACTCGGCAAGTTTGTTCCGACTGTAACTCCGGGGATGAGGAACGGGG 110 cg11601252 15 68122139 LBXCOR1 CACAGAGGAGGGGTTGGGGGCAGCGGAAAATCGGGCAGGTCGAGGCAGCCGAACCCCGGA[CG] 110 ATGTCCCCCCACCCACCCCGAAGGTCGCAGCCTGGGCCGCGTTCTCAGCAGGAGTCGGGC 111 cg10698928 8 65290320 MIR124-2 CAGAAGGACCATCTGCGGACTCGTTTTCACTGCTCCAGCTCCCGAAATCGTTTCTCTGGA[CG] 111 CGGCAGTATCCGCAGCGCATGCACCCTCCTCTGGACTTCCGCAGCCCAGACCTGCGCTTA 112 cg25832771 8 72756058 MSC GGGGGCACCCAGGGCAGGCTGGTCTTGAGCCTGGAGAAGGCTTTGCTCAGCACGCGCATC[CG] 112 GGCACGCTCACGGGCGTTGGCCGCGTTCCGCTGCGACTGCTTGCACTCTGCGGCTGAGCC 113 cg18507379 5 140787507 PCDHGA4,  TCGGAGACCGAATTCAAAATGAAAAACCGGGCTGCTGTCCCGCACGGAGCCTCTGGGCGC[CG] 113 PCDHGA6; CTGTCGGCCAGTGCAGAGCAAGCGCTGACGCCGGGGATCCGTCAGCCTCTGGCCTGGGAT PCDHGA9; PCDHGA1; PCDHGA8; PCDHGA5; PCDHGB1; PCDHGB6; PCDHGB4; PCDHGA3; PCDHGA2; PCDHGA7; PCDHGB2; PCDHGB6; PCDHGB5; PCDHGB3 114 cg14733048 8 109095782 RSPO2; CTTTCTCCACGGTCACTTCACAGCTAAGATTTCTTTCTTTCCGAGCTGTAGAAGGCAGAA[CG] 114 RSPO2 CTCTCGGGAGGACGAAGTGATCCGAAGGGATGTGGCAAGCGCACTTTCCGATGGAGATGC 115 cg09735723 10 106402042 SORCS3 GGGAGGGATCTGTGCTCACTTTCTCCAATACTTGGCTTGGAGGGTCAGTTTTCCTGTTTG[CG] 115 GGGTGCTTGAATTCTTGGATGAGAAAAAGGGCTGACTTGGGGCGGGAGCCGCTGAACAGA 116 cg05099508 10 22634432 SPAG6; SPAG5; GCAAGACGGCCGCGTTCCGGTTCCGGTAGGTTGCCCGGGAGACGCGGGTACACAGAGAAG[CG] 116 SPAG6; GCTCCCGTCGGAGGCCGAGTCGTCGCCACGATCGCCCCCTTGGTGGACTCGCAGGCCGAG SPAG6 117 cg14638883 6 166582201 T TCTGAGAAGTGTCCTCCTCGCTCTCTTATAAAAACAGGACTTGTTGCCGAGGTCAGCGCG[CG] 117 CATCGAGTGTGCCAGGCGTGTGCGTGGTTTCTGCTGTGTCATTGCTTTCACGGAAGGTGG 118 cg26673012 7 35293753 TBX20; GGAACTACGGACAGTGAGCCCTGGCGCTCGCTGCCCTGCGCCTTAATTTGCTGGCGGCGG[CG] 118 TBX20 ATCCCGGAGGCCCGCAGCCAGTCAGCGCCGTCTCACGTCACCGCTTCCTGATTCCGCCGC 119 cg14763548 20 25052447 VSX1; VSX1 GCTCGGGGCCCCTGGGCGGCAGGAACGGCACGTCCGCTAGGAGCAGGCAGGGTGCTCGAG[CG] 119 GCCGCCGGCGGCTGCGTGCCGAAGCCACAGAGGAGGCCGAGTCCCAGCGGTAGGGCCCCA 120 cg22884656 2 45157296 AATCACTGTAGTTAAAAATGTATGGGATTTTTGCCGTCGGAGCACCTCGTATCCGGCGCG[CG] 120 GGCCCAGTGTGGGACTGCGGCTGGGAGCCCGGGCCCTCCGGGAGTGAAGATACCTTTGGA 121 cg21401879 2 45162036 GCGGGCTCCCCCTGGCCACATCCCGGGCCTCTCACACAAGAAGAATAGTTCTGTTTCCGC[CG] 121 TAAACCCCCACACAAAGGCTGCCCGGCCACCGGGTCCCCTGTCCCCCTTCCCGGGGACTT 122 cg09797577 6 28778226 GTAGGCTGGACCAGGAAGGAGACCTGGTTCGTTTCGCCCAGGCTGTCACGGCTTCAAGAG[CG] 122 CCTCTCCGCTATTTCCGTCGCTCGACAGACGGGCTGAGCTCTTTGGAGTGATGTTGGGTT 123 cg20567847 7 155167038 CCGGCGGGGGCTGGGCCGGGCGGGTGGGTTTCTGAGCCGCAGCGCTTGGAGCTGGGGGAG[CG] 123 GGAGCAGGGGCGGCCCGGCGGGCGGGCCGGGACCCGGCTTTTCCGGCTACCCGTGGGCCA 124 cg04828133 7 155259845 GGAGGTTAATAGCGACTGACGACAAAGGGCCAAGGTGCAATTCCTAAAGCGGGGATTCGC[CG] 124 GGTGAGGCAGAAATCAGCCTCCGGGGAGATGGGTCCCCCCTTCCCGACGCGCCCCTGCAC 125 cg04521510 7 27242044 CTTCGGTTTTCCGGCCAGACCCGGAAAAACGAAAACACAGCTTGGGGAGCCCCCACTAGC[CG] 125 GCGCCTGTGCCAGCTCACCTCTGGCCATGGCGCAGCTGCCGGTGCACACGGCGGCCAAGG 126 cg24304093 11 14926738 TCGGCCTGGCCAGGCGGGGGGTCTTCTTCCCAGTCCGCGGAAGCCAGGTCCGGGAGACGG[CG] 126 GGGCTCACTGTGCCTTCGGACCTACACTCTGCTCGGGGGCCGGATTTCTGCAAAGCGTCC 127 cg24292235 12 8171463 AGCCTAGGATTCGACTTTGAATGGTCCGTTAATGTGGTCGCAAAACGTGACTCGGTTCAT[CG] 127 GGCGCTCCCTGTAAGCAAGACAAGCACCCACCTGCGGTCAGAGCAGGGGTCCGGCTCGCG 128 cg25092681 5 16180033 Mar-11 GCGGGAGGGAGGAGCGGCGCGGCGGGGGTGACGGGGCGCGGGCGCGGGGTGGGCTGGGGG[CG] 128 CGGATCAGTGGGACGGAGTTCGGGGTTCGGCTCCGAGCGGGCGGGCTGGAAGTGGGGGAT 129 cg02984514 17 31618409 ACCN1; GGGGGCGGAACACCAGGCCTCCTGACTGCCGGGGAGTGGGGCGCAGAGGGAGCGGGTTCG[CG] 129 ACCN1 CGGAGGGCAACTCACGGAGGAGAAGTTGTGCGGCCCGCAGAGCTCGCCGCGGTACTTGCA 130 cg07113542 20 2781262 CPXM1; AGCAGGAGCCCCCACATGGCGGGGATTGAGTGCCAGGGGCGCGCGGGCTACGGCGGGTGG[CG] 130 CPXM1 GGTCGGTCTCTTCCTGCCGAGTGCGCCGAGCCCCCCGCCCCTTCCTGCCCCCCGCCCCTC 131 cg04609163 11 125774090 DDX25; CGTAAAGCGCGGCGGGGAGTCCGGGGGGCTCCCGCCTGGAGGGCTGTGTGAGCGGCGGGC[CG] 131 PUS3 CGGGGCGGCGCGGGGGGCGCTCTCCACTCTGCGGAAGCTGCCCCCTCTGCCCTCCGGTCC 132 cg11193865 14 101193038 DLK1 CGGCCGCGTGCTGTACAGTGTGAGGGAACGTGTACCAAACGCTCGCGGGATACCTGTGCC[CG] 132 TCTAGCCAAGAGTGCACCCGTGTGCGCGAGCGGGCTTCTGGGACGCCGCCGTGGTCGGGG 133 cg09124223 7 153749738 DPP6; DPP6 GGGGAGGGGGCGGAGGAGGCTGAGCCAGGCAGAGTCGCCAGCGGAGACTCGCGAGTGGCG[CG] 133 CGGGAGGAGCGGCCGCCGGCGCTGGGCTTGCCTTGCTGCTGCTGCTGCTGCTGCCTCCCC 134 cg14768785 3 172166517 GHSR; GHSR ACACCTCCCCTTTCCCCCACCAACTCCCCCAAAGTTTCTCCCAACACATCCTCCGGCCGA[CG] 134 CCCACACGCATACCTGTCACCAGCCCTGCCTCGCATTTGCGTTCTCGATCCAGTTCCATC 135 cg12477716 17 47073436 IGF2BP1; GTATCCGGGACTCCGAAACGCGCGGCGAGCAGCCCCCTCCCCCACCGCCCAGACGGGGTG[CG] 135 IGF2BP1 ACCGCCCACGTGTCGCCCCTTGCCCAGTCGGGTCCTTCCCTCGGGCTCCGGGAGCCGGAG 136 cg24826867 16 85932853 IRF8 GAACGCGGGCGGCGAGACGGCGGCAGGACGGCGGCAGGTAGGCACAGTGGGCGGGTAGGG[CG] 136 CCCGTGTCCCGCGCGGTCCGGTCCCGCGGGGTCCCCGACGCCAGGCGGGGCGTGGGGGTG 137 cg24663256 4 21950307 KCNIP4; ACATTCATGTCTAGGGACGCAGGGTGCAGAAGCGAGACTCGAGAGTCCACCGGCCAGGGG[CG] 137 KCNIP4 TCTGTCCACGGGTCTGCACGGGAGCGCACCGCCGCTCGGCCCGGGGGCGTCCGTGGCGCT 138 cg06653045 17 68165064 KCN12 TAATTCGCGAACAGTCGGGAGAACAAACAGCCAAGCGGCGCTGCAGTGGCCGCACTTGCG[CG] 138 CGTCTCAATCCTGGGGGCTCTGCGCGCCCGCCCCAGTCCCTCGCCCCATTGACTCAGTGG 139 cg13592399 14 52535758 NID2; NID2 CCGGTCCCCCTCCATGCTCGCTCGGCCGTGCGCTTACCCGCTGCACAACGCGTCCCGCCC[CG] 139 GCCTCCAGCCCACTCTCCGCGCCGCGCCAGCCTCGAACCTGGATCTCCGCGGGCGCCTGG 140 cg20008332 2 5833169 SOX11 CGCTGGAAAATGCTGAAGGACAGCGAGAAGATCCCGTTCATCCGGGAGGCGGAGCGGCTG[CG] 140 GCTCAAGCACATGGCCGACTACCCCGACTACAAGTACCGGCCCCGGAAAAAGCCCAAAAT 141 cg02071005 2 175200844 SP9 TTCCTCTTGCCCTCCTCCTTCTTGCTCCCTCCCCCATCCCACCCACTCTAGGAAGAGCCG[CG] 141 CTTCGGAACGACCCCGTTGGCCATGCTGGCGGCGACCTGCAACAAGATCGGCAACACGAG 142 cg23302682 6 166580952 T ATCATCTCATTGGTGAGCTCCTTGAAGCGCAGCCACAGCTCGCTCTCCTCCAGGCCCACG[CG] 142 CAGTTCGCGCTCTGTGGGGTCGCCCTTCTCGCTGCCCGCCTGCAGCTCATTCTCCACGGC 143 cg07978472 4 122686493 TMEM155; GCCAAGGAGCTGAGGAAATCCGGCGCAGACTCTCCCAGCTGGCACCAAAGCCTTCCGCTT[CG] 143 LOC100192379 CCGAGATCCTCTCAGGTGCTCTTGAGGACGCGAGCGACTTCCCTAGGAGCGAACTTCCGC 144 cg14732324 5 528621 CCCTCGGGGACAGCCCGGCCGGCCACGCCGCCGAACTCGCCATGGGCCTCTCTCCGCATC[CG] 144 CATGTGCATCCGCAACCGCTTCCGTCCCGCTGAGCGCACGAACCCTCTCGCTCCTGTCCC 145 cg00228475 6 58149279 TCCTCAAGAGAGGTAGGGTCCGTTCCCCCCGGCGGGGCCGGTTAGCTCAGTTGGTTAGAG[CG] 145 TGGCGCTAATAACGCCAAGGTCGCGGGTTCGATCCCCGTACGGGCCACAGGCTTTTCTAA 146 cg12812583 8 23567310 GAGCAGGCTCCCCAGCGTAGCGAGTCCTTGTTATCGAAAGGGTCGTTTCGGCTCAGGATG[CG] 146 CGCTCCCGGCGTAGACCTGGGGATAGGGGTCCCTGTCCCGCTCGCCCCACCCCTGCAGGG 147 cg01163842 14 95235125 GSC GTGGCGGCGGGACACCCCGCGCAGGCCAACAAAAGGAGGGGAGCCGCTCGCTCCCGCTTC[CG] 147 CGTTTTCATTCAACTTCCTGGGCCTAAAGCGCCCTCCAGCAGCCTGCGGGCCGCCATCGG 148 cg17394649 6 29760164 HCG4 AAACGCCGTCTGTGGGGAGTAGCTAGGGGCCTGCCCGGCGGGGGCGCAGGAACCCGGTTG[CG] 148 CTGCCGGGAGGAGGGTCGGGAGGGTCTCAGCCCCCTCCTTGCTCCCAGGCTTCCACTCCT 149 cg10723962 6 26240782 HIST1H4F GCGTGACAACATACAGGGCATCACGAAGCCCGCCATCCGTCGCTTGGCCCGACGCGGCGG[CG] 149 TGAAACGCATTTCGGGCCTCATTTATGAGGAGACCCGCGGTGTTCTTAAGGTGTTCCTGG 150 cg17466857 7 27225528 HOXA11AS; GGCTTGTCCGATTTGCACGGTGACTTGATTACACTCTCTCATTCATGGTCACTTCCGAAG[CG] 150 HOXA11 CTTTAGTGCCTTCCGTCCCTAAACCGCCAACAGCCAGAACGGCTTCTCCCCGCGGTTTGT

TABLE 2 Table 2 below provides exemplary primers which may be used to amplify or sequence MVPs defined in Table 1 above. Antisense Sense Chr Sense Start Start Sense sequence SEQ ID NO 2 119, 606, 705 119, 606, 804 TTGGAGGATTAGTATTYGATTATGTTGAA 152 19  54, 926, 752  54, 926, 852 CCTCAACTTAAATACATCTCCTCC 153 7  70, 597, 038  70, 597, 139 GGGGTGGGGTGGATTT 154 4 111, 544, 332 111, 544, 433 CCTCCTCCCAAACCCTTCT 155 7  27, 205, 053  27, 205, 155 GTTTAGGGTTTTAGTGGTGGT 156 7   5, 569, 082   5, 569, 184 GGAAATYGGGAGGTTTTTGTGTA 157 1 237, 205, 102 237, 205, 205 ATCTCTACTTAAAAATTAACCACCC 158 15  68, 122, 063  68, 122, 166 RCCTTACCAAATTCCTCACAA 159 13  78, 493, 251  78, 493, 354 TTTGGTTGGTTGAGTTATAGTTTT 160 7  70, 597, 035  70, 597, 140 GGYGGGGTGGGGTGGA 161 8  65, 290, 282  65, 290, 387 RTTTTCACTACTCCAACTCCC 162 18  74, 961, 907  74, 962, 016 TTTGGAAAAGTYGGGAGGGAG 163 6  28, 367, 508  28, 367, 619 YGGAATTTATTAAAAGTGATTTATAAAGGT 164 2 223, 163, 755 223, 163, 866 CCCAAAACTTAATCAAAAACCCT 165 6 166, 582, 153 166, 582, 264 CCTCCTCRCTCTCTTATAAAAACAAAACT 166 8  26, 723, 555  26, 723, 669 GGGGAATTTTGGAGGATGTATT 167 6 108, 440, 301 108, 440, 416 GGGGATTAAGGGATTGTTTATTTTG 168 8 132, 054, 523 132, 054, 639 GTTTAGTGTYGGTGTTAATGATAGATG 169 5 115, 152, 343 115, 152, 460 AGGTTTAGATTTGTGGGGTTTA 170 10 106, 401, 968 106, 402, 085 TTAAAGATGAGTGGGGGAGG 171 11  20, 618, 161  20, 618, 279 AAAGTAATTAAGGTYGTAGTGATTGGT 172 19  54, 926, 741  54, 926, 860 GGGTTATYGGTTTTTAGTTTGGGTG 173 2 223, 163, 760 223, 163, 880 AGTTTGGTTAGGAGTTTTGAGT 174 22  22, 862, 820  22, 862, 940 CCTACACTCCCCCACAAAA 175 11 120, 434, 955 120, 435, 076 YGGGGGAAAATGTTTTTATTGA 176 8  23, 563, 877  23, 563, 998 CCTCACAAAAACCCCCAAAA 177 2 198, 650, 902 198, 651, 024 TATTTGGYGGGTGGGGAGAA 178 6  10, 882, 276  10, 882, 398 ACCTCTAACAACTACCCCCT 179 20  25, 065, 180  25, 065, 302 CCACCCAAAACCCAACRTCAAATTA 180 7  27, 204, 952  27, 205, 074 GTTTGTYGGGGAGGTTGGTTT 181 5 172, 671, 485 172, 671, 608 CCTCTTCCTTCCTTCCAAATC 182 3 147, 113, 701 147, 113, 826 GGGAAAGGATAAGGGAAGGG 183 12  85, 667, 301  85, 667, 427 AGAGTTGTTTTTGATTGTAAGGGA 184 7  27, 232, 754  27, 232, 882 ACCACTCACCCRCACAAACAC 185 8 145, 106, 354 145, 106, 483 RTATAAAAACAAAACACATCCTATTAAC 186 11 112, 833, 711 112, 833, 841 GGGGYGGGTTAGGGAGTATTT 187 7 155, 259, 749 155, 259, 880 AAATTGTAGTTTGTTAGGTTGAGT 188 4 174, 450, 311 174, 450, 443 AAAAATAAAAATTCTCCTCATaactacaa 189 8 132, 054, 519 132, 054, 651 AAAAACTTAATACCRATACTAATAACAAATAAAC 190 10 118, 030, 953 118, 031, 086 AACTCCCRAACCACCCCC 191 1 111, 217, 615 111, 217, 749 CTCTCCRCCCCCTCCCCTA 192 15  86, 233, 181  86, 233, 315 CTCAAAACTACTAAACTCTACCCTA 193 1 248, 020, 537 248, 020, 671 GGGAGGYGGGTTATGGTTTGG 194 2  66, 667, 006  66, 667, 141 TTTGAGAAATGTGAATTAGTTATTTGT 195 5  16, 180, 236  16, 180, 371 TTGGATGGGTYGGTGGGATG 196 3  42, 306, 888  42, 307, 023 ACCACAACTAACTCTTAATATCCT 197 7  24, 323, 740  24, 323, 875 AACTACCCRAAACCCCTCCT 198 10  15, 761, 845  15, 761, 980 AAAATCTAACRATCCCCAACTACC 199 8  72, 755, 998  72, 756, 133 GGGGGTATTTAGGGTAGGTTG 200 12  54, 408, 585  54, 408, 721 YGTTTGGAAAGAAATGGAGGTA 201 3 147, 113, 681 147, 113, 817 CAAAAACCTTCCCACACCC 202 11  71, 955, 309  71, 955, 445 AAAACCCAAACACCRCCCAAAA 203 6  29, 760, 051  29, 760, 188 GGGGGTTTTGGTTTTGATTTAG 204 2  66, 667, 480  66, 667, 618 AAGATTGAGTTTTGGGTTTGTT 205 11  71, 955, 292  71, 955, 431 GAAGGGGYGGGGTTTAGGAG 206 1  50, 886, 838  50, 886, 977 TTCTCCTCCTACRCCTACTACCT 207 14  95, 235, 085  95, 235, 226 GTAGGTTAATAAAAGGAGGGGAG 208 7   5, 569, 280   5, 569, 421 ATTATTTATGGTGAGTTGYGAGAATAGT 209 10  15, 761, 835  15, 761, 977 GGGGGTTGGTGGAATTTG 210 1 119, 530, 560 119, 530, 702 TCTCCACCCTCCCCCT 211 4 174, 450, 301 174, 450, 443 AGTTAGTTATGGAAGTAGGGGT 212 7   5, 569, 274   5, 569, 417 GATATTATTATTTATGGTGAGTTGYGAGAATA 213 2 198, 650, 997 198, 651, 141 AGTTTYGGTTGTGGTTTTGGGA 214 3  42, 306, 941  42, 307, 085 GGATGATATTATTTGGTTTTGTTTAGAG 215 7  19, 184, 912  19, 185, 056 GGGGAGGTTAGGGATAGGTT 216 14  52, 734, 499  52, 734, 643 CAACCTTCACTCCAACCCT 217 13 109, 147, 820 109, 147, 965 GGGGAAYGTGGAAAGGAGGG 218 1  50, 513, 833  50, 513, 979 AAAAATACRCCTCAAAAACCAAATAAAAAC 219 14 101, 192, 985 101, 193, 133 GTGTTGTATAGTGTGAGGGAA 220 20  21, 686, 229  21, 686, 377 RAAAACCAATAAACTACCAACTC 221 7  24, 324, 907  24, 325, 055 GTTTTTGTTYGTGTGTTTGGGTG 222 2  66, 666, 426  66, 666, 575 TGGGGTTGGGATTTTTAGGT 223 5  76, 923, 868  76, 924, 017 GAGTTTTGGTAGGTGTTGGT 224 2  66, 667, 385  66, 667, 534 CTCTACAAAATAAAATCAAAAACACCA 225 10 106, 401, 990 106, 402, 139 TCTATACTCACTTTCTCCAATACTT 226 20   2, 781, 198   2, 781, 347 AACAAACAAAAACCCCCACA 227 11 112, 833, 653 112, 833, 803 CAACTCCAAATACCRTTATACCTACCCTA 228 1 248, 020, 634 248, 020, 786 TGGATTGYGGTTATAGTTTTTGTTTTAGG 229 13  78, 493, 250  78, 493, 403 CCCTAACTAACTAAACTACAACTCC 230 6 108, 440, 232 108, 440, 386 TCTAAAAATTAACTCTAACTTCCCCA 231 2 182, 322, 443 182, 322, 597 TGTTGTTGTTGTGTTTGGGG 232 11  20, 618, 126  20, 618, 280 RAAAACAAAATCAATTACTATTTTTCATCT 233 8 145, 106, 394 145, 106, 549 GGGGAGGYGGGTTTAGTGTAG 234 15  86, 233, 182  86, 233, 337 TTAAAGTTGTTGAGTTTTGTTTTGA 235 6  29, 760, 062  29, 760, 217 TCCTAACCCAAACCTAAACAAA 236 12  85, 667, 262  85, 667, 417 TCAAAATAAAAACTCCTCCACCTAT 237 4 147, 561, 686 147, 561, 841 TGTAGGTATGGTTYGAGGAGGT 238 6  28, 778, 138  28, 778, 295 GTGAGGGAAGAGAGGTGTTT 239 7  27, 225, 023  27, 225, 180 TGTAGTTATTTTAGGGGAAGTAATAGAT 240 14  24, 045, 512  24, 045, 669 GGTAGGGGYGGGGGTGG 241 13  28, 366, 707  28, 366, 864 RTTCAACCTCCTAAACAAAAACAA 242 4 147, 561, 678 147, 561, 835 ACCCATCCTACAAACATAACTC 243 10  22, 518, 210  22, 518, 368 GAAGATTTYGGGGAAGGAGTGG 244 7  27, 225, 011  27, 225, 169 ACTTCCTTTCTTTATAACCACCTC 245 14  95, 234, 999  95, 235, 157 RAAAAACAAAACAATTCAAATCAA 246 1  50, 513, 832  50, 513, 991 GGAAGATGYGTTTTAAGAATTAGGTAGAA 247 10 106, 401, 430 106, 401, 589 GGGTYGGGGTATTTTAGTTTTTGT 248 X 136, 656, 476 136, 656, 635 GAGGTTTGGGGATTGGTTG 249 2  66, 666, 980  66, 667, 139 CCCTCTTCCCTCTCTTAACAC 250 10 106, 401, 346 106, 401, 505 AAAACTACTACCCCAACAAAAC 251 12  62, 584, 934  62, 585, 094 GGGGAAGGGAGAYGTGTGTA 252 1 111, 217, 518 111, 217, 678 CCCACCRCCTATTACAACCAAAAC 253 2  66, 666, 422  66, 666, 582 ACCTTAAAATTAAAATCCCTAAATACAAC 254 5 115, 152, 387 115, 152, 547 RCTTAAAATCACTAAAAATATACCAAC 255 5 180, 596, 604 180, 596, 764 GTGTAAgttttygtagtgtagtggt 256 22  50, 216, 544  50, 216, 704 TCCCAAAATCCCACACTACA 257 7  27, 225, 490  27, 225, 651 ACTTAATTACACTCTCTCATTCATAATC 258 11 120, 434, 988 120, 435, 149 RCAACTTCTACCTTTTATTACAAAC 259 1 237, 205, 044 237, 205, 207 AATTTTTATTTGATTATGAATAGAGGTAATTT 260 20   2, 781, 222   2, 781, 385 GGGGATTGAGTGTTAGGGG 261 7   5, 569, 852   5, 570, 016 CCACCCTACRATCCCCATTAAC 262 2 175, 199, 604 175, 199, 769 TCTACTCTACCTACRCCCTCATTAAA 263 18  44, 787, 470  44, 787, 635 CRAAATCCTCCCTCCTACCTC 264 11  14, 926, 608  14, 926, 773 CACAAACCCAAAAACCCCA 265 20  25, 062, 422  25, 062, 588 GTTAGGAGTAGGTAGGGTGT 266 6 391, 841 392, 007 GTGAGGTTGATATTAGAGAGGAT 267 7  19, 184, 852  19, 185, 019 RCAAAAACTAAATCCCCCAAAAA 268 5 140, 787, 404 140, 787, 572 TTGAGGGGGATGTATATTTGTATT 269 18  74, 961, 845  74, 962, 014 RACTCTATCCACCACCAA 270 6  27, 513, 424  27, 513, 594 AACCATTATAAATTCACCCCAAC 271 6  26, 240, 675  26, 240, 845 GGTAAAGGTTTAGGAAAGGGAG 272 6  28, 778, 185  28, 778, 355 AAACCTAATTCRTTTCACCCAAACTATC 273 2  66, 666, 605  66, 666, 776 TCCACCRAATCCTAAATATACAATAAAAA 274 7  27, 204, 213  27, 204, 386 TTAAGGAGAGGGGYGGTTAGTT 275 20  25, 065, 065  25, 065, 239 ACATTTTCATAACCTCCTACAATAAA 276 7  20, 817, 774  20, 817, 949 CTACAAAACRAAAACAAAACACAAAATAA 277 11 125, 773, 960 125, 774, 135 AACCAATTCRCTAACCTTTCTAACATC 278 7  27, 204, 899  27, 205, 074 CCTTAAACTAAAAACTACACRAACTAAAAT 279 5 172, 671, 421 172, 671, 597 AGTGTTGAATTGATGTTGGAAA 280 18  55, 108, 733  55, 108, 909 TCTACACCCCCTCCCC 281 14  70, 653, 838  70, 654, 015 TTTTTGATTATTTAGGAGTTTGGTTG 282 6  99, 841, 571  99, 841, 748 AAACACAAATACAAAACTATACATAACT 283 14  24, 045, 441  24, 045, 620 CCCCRAACCCAACCAACA 284 2  66, 666, 618  66, 666, 799 GGATGTGTAGTGGAGGGG 285 10 135, 043, 371 135, 043, 552 gggggtAGTATTGGGGGT 286 4 122, 686, 466 122, 686, 647 CCCAACTAACACCAAAACCTT 287 20  37, 352, 962  37, 353, 143 TCCAATCCCCCACCCC 288 7  27, 204, 196  27, 204, 378 ATCCTAAACCTAAACAACCAAAAA 289 8  26, 723, 434  26, 723, 616 CCAAAACCCCAAACTCCAA 290 1  50, 886, 642  50, 886, 825 RCTCTCACCTAAACCCCC 291 6  27, 513, 336  27, 513, 519 GTTATTGATGTGTTTTGAATGAGTT 292 7   5, 569, 687   5, 569, 870 ACACCCCRAACCCCAAAAC 293 10  22, 518, 180  22, 518, 364 CCAACACCCTAACRAAACCTAAAC 294 7  27, 232, 735  27, 232, 920 GTTAGTTTGTAGTTGYGGGGTTATTTA 295 2 162, 283, 572 162, 283, 757 ACAATACATTCRTATCATCACCCACC 296 2  45, 161, 903  45, 162, 088 aaatctaccccaccctacc 297 6 166, 582, 063 166, 582, 250 GTTTTTATTATTTGGAAAAGGAAGGTT 298 8  72, 756, 006  72, 756, 193 CCAAAACAAACTAATCTTAAACCTAAA 299 14  29, 254, 632  29, 254, 819 CCCTCTTTCCCCTTCAATCTT 300 15  76, 629, 983  76, 630, 170 ATCATCTTTCCTTATAATCRCAAATCTTAAAAA 301 8  72, 754, 933  72, 755, 121 ACTTCTTTAATATCAAAATCCRATCTTCC 302 8  23, 567, 163  23, 567, 352 atactttctaaCCCCTCTCRAAAAATA 303 12  62, 584, 867  62, 585, 056 ACCCRCATCTACCCTCACCT 304 7   5, 569, 805   5, 569, 994 CAATAACRCCCCAACCCCCAC 305 19  58, 238, 678  58, 238, 868 CTAAAACACAAAAACTACAAACACT 306 5  16, 180, 017  16, 180, 207 yggggtgggttgggg 307 13  28, 366, 672  28, 366, 863 AGAGGTTATTGTTTTAGTTTAGGTTT 308 1 248, 020, 475 248, 020, 666 ACTCCTCCCCCTATACAAAC 309 4 147, 559, 282 147, 559, 473 AAACCCTACAACCCCTCC 310 3 147, 113, 702 147, 113, 894 GGAAAGGATAAGGGAAGGGT 311 4 147, 559, 273 147, 559, 465 AGTGAGAGAAAGTTTTGTAGTTTT 312 6  10, 882, 217  10, 882, 409 GGGYGGGGTTTTGTTTTTGGT 313 12  54, 408, 539  54, 408, 731 ACAATACCCRCTCCCCCACC 314 18  22, 930, 230  22, 930, 422 RATCAACTTCTAAAAATAACCAAAAA 315 8  72, 754, 903  72, 755, 096 GTGGTTTTATATAGTTTGTTGGTTG 316 2  66, 667, 490  66, 667, 683 TCTAAACTTATTTTCAACTTATTTAATTCAC 317 20  21, 492, 888  21, 493, 082 GATGAGGTTGGTTAGGTGTT 318 6 391, 785 391, 979 CCCTCAACTCCRAATCCAAAAC 319 7  24, 324, 845  24, 325, 039 CTTCTATACCTACAAATACTAAATAACAAA 320 7  70, 596, 935  70, 597, 129 AAACCRATATACCCTAAAAACCCC 321 5 528, 491 528, 685 RACAAACCCTCCCAACA 322 2  66, 667, 301  66, 667, 496 TGTTTGAGTGTTTGTTTGTAGAT 323 4 174, 450, 245 174, 450, 440 GGTTGTAGGATAGGGTTATGTTG 324 20  21, 686, 179  21, 686, 374 GGAGGGGGTAGAGTTTTAGG 325 1 149, 719, 383 149, 719, 579 ATGTTGAGGGTGTTAYGGTTTTATT 326 7   1, 272, 352   1, 272, 548 GTTTATTGAGGTGTATGTTAGGTATAAT 327 2 119, 607, 734 119, 607, 930 AATTCATTTTACCTTTCATATAATAAAACC 328 15  68, 122, 089  68, 122, 286 GGGTTGGGGGTAGYGGAAAAT 329 15  76, 630, 037  76, 630, 234 GTTGGGTTGGGGTTGGA 330 3 129, 693, 494 129, 693, 691 CCTCCCCACTAACCTCAC 331 5 172, 659, 564 172, 659, 761 TCCCTTCCCTACCAAACTC 332 7 149, 917, 234 149, 917, 431 CCCTCCACAAACCTACCAAA 333 14  60, 975, 848  60, 976, 045 CCAATCCRCCCACCCCAATAAC 334 2 162, 283, 566 162, 283, 764 AGTGGAGTAGTGTATTYGTGTTATTATTTA 335 20  21, 081, 785  21, 081, 983 GGGATGGGGAAATTATTTGATTAG 336 8  23, 563, 886  23, 564, 084 GATTTTTAGGAGGTTTYGAATTATTTAGTTT 337 16  85, 932, 830  85, 933, 028 GTAGGTATAGTGGGYGGGTAGG 338 13  53, 420, 219  53, 420, 417 CCCCTTTACRCACCTCCTTCTT 339 2 119, 606, 711 119, 606, 910 AACCAATATCCRACTATACTAAAACCTA 340 7  27, 205, 063  27, 205, 262 CCAATAATAACCATCACCRTACCCAA 341 22  50, 216, 594  50, 216, 793 TTTATTTTTGYGGGGGAATATAAGGAG 342 22  50, 922, 498  50, 922, 697 CAACAAAACTCAAAACATTCCC 343 22  22, 862, 796  22, 862, 997 GGGGATYGTGGGATTTGGTT 344 1 111, 217, 055 111, 217, 259 GGGAATTGGTAAAGGGTTTTTAG 345 6  28, 367, 514  28, 367, 722 CCATCAAAAATAACCCACAAAAAC 346 1 111, 217, 355 111, 217, 565 GGGYGTTGAGGAGGGTGG 347 2 119, 607, 767 119, 607, 977 ATTTGTTATAGTAATGGGATGATAAATG 348 7 149, 917, 184 149, 917, 394 AGTTATYGATTTTTGTAAGGGATGTAGA 349 2  63, 281, 084  63, 281, 295 AAGGTYGAGATTTGGGTTTGTTAG 350 1  50, 886, 613  50, 886, 825 TTTGTTGTTTTTATTTGYGGTTTGGT 351 5 135, 266, 111 135, 266, 323 RAACTCCAACACCTTAACC 352 7 155, 166, 900 155, 167, 114 GGTAGGAAGAAGGGYGATGTTTTT 353 14  95, 239, 509  95, 239, 725 ACTAAACAAACCATCAAAACCC 354 10 118, 030, 867 118, 031, 084 TTTTAAGGGTTTGTTTTAGAGTTTG 355 3 147, 113, 558 147, 113, 776 AGTTGGGATTTGAGGAAATTTAG 356 11  20, 618, 134  20, 618, 352 AATCAATTACTATTTTTCATCTTTAACAAAA 357 10 118, 030, 866 118, 031, 086 ACTTCAAAAATTTACTCCAAAACCT 358 11  71, 955, 207  71, 955, 427 AATACAACRCAAATACAACCAACCC 359 11  14, 926, 603  14, 926, 824 GGGTGTATAGGTTTAGGGGT 360 5  15, 500, 202  15, 500, 423 CCTCCCCRAAACTCCAACTAT 361 2  63, 281, 053  63, 281, 282 TCTTTTATTCCCAATTCRACTTTCTTT 362 14  29, 254, 647  29, 254, 879 AGTTTTTAGGGAGGGGGAG 363 1 111, 217, 544 111, 217, 780 YGATGTTTTGTTTGGGTTTGG 364 11  31, 848, 615  31, 848, 857 GTTGGGTTGGGAGAAGTTT 365 4 111, 544, 173 111, 544, 425 GGGTTTTGTYGTAGTTTAGTTTTAGG 366 7  27, 205, 054  27, 205, 314 TTTAGGGTTTTAGTGGTGGTT 367 19  58, 238, 712  58, 238, 972 GGAGTYGGAGAAAGGGTGATT 368 5 180, 596, 452 180, 596, 712 CATCTCAACCTTCCAAATACTAAA 369 2  63, 283, 871  63, 284, 132 TGGYGTGTTTTTGTTTATTGGAGTATT 370 15  65, 116, 211  65, 116, 473 ACAATCCRAAAACAACAACTACACT 371 5 140, 787, 349 140, 787, 617 ATTTAAAACACAAAACATAAAAATATCTACTA 372 14  95, 239, 425  95, 239, 697 GGAGGTAGGTTTYGGGAAAGG 373 10 135, 043, 275 135, 043, 552 ACCCATCCCCTAACCTAAC 374 19  58, 238, 625  58, 238, 903 CCCACACCCACCCAC 375 6  26, 240, 714  26, 240, 993 AAAATACTACRTAACAACATACAAAACATCAC 376 20  25, 065, 005  25, 065, 285 TGTGTTTTGGGTTATTTTGTGTT 377 7 155, 259, 636 155, 259, 918 AAAATTCTACTAAAAATCATTCTATCTCC 378 5 172, 659, 477 172, 659, 760 GGATTTAGGGTTATGTTGGGAG 379 7  20, 817, 773  20, 818, 057 GTTGTAGAAYGGGAGTAGGGTATAG 380 14  52, 734, 368  52, 734, 652 GGAGGTTTTTGTYGTGGGGAATA 381 20  61, 638, 444  61, 638, 729 CACAAACCRAAAATAAAAACTCTAAACC 382 18  77, 547, 903  77, 548, 190 CTAACAAAACRACCCAACCAAAAA 383 11 120, 434, 917 120, 435, 205 TTAGAYGGTATTAGGTAGTTGAATTTAGT 384 9  37, 002, 424  37, 002, 713 GGTTAGTAAGAATGTTATAGTTTTATTTTGT 385 1  91, 182, 909  91, 183, 198 AACAATCTATAAATACTTTCRACACAACT 386 18  44, 787, 350  44, 787, 639 AAAACTAAAAATTCCTAAAATCCCTTTA 387 1 111, 217, 246 111, 217, 537 CAACAAATAATCCCCRAACACCA 388 7   1, 272, 400   1, 272, 691 RTTTCCCTACACCCAACAC 389 11 125, 774, 055 125, 774, 347 GGGTTTTYGTTTGGAGGGTTGT 390 7  24, 323, 635  24, 323, 929 GGGGTGTGGGTGGTTTT 391 X 136, 656, 432 136, 656, 726 RACCCCTCCAACCTTT 392 1  91, 182, 902  91, 183, 197 GGGAAGTGGTAATTTGTGGATA 393 7  27, 225, 460  27, 225, 755 TGTTTGGTGGTTTGTTYGATTTGTA 394 2 182, 322, 235 182, 322, 530 CTTCCCTCTCTCCTTCCTTTA 395 2  63, 283, 941  63, 284, 236 CAAACTACTACCCCTTCAACT 396 11  20, 618, 153  20, 618, 450 TTTTTGATAAAGTAATTAAGGTYGTAGTGA 397 14  70, 653, 836  70, 654, 133 ATTTCTTAACCACCCAAAAACTTA 398 1 119, 530, 348 119, 530, 646 ATTAAGGTTTTAAYGGAGAAGGTATGT 399 17  47, 073, 268  47, 073, 566 CCCCTAACRCCATCCCAACC 400 8  23, 567, 042  23, 567, 341 GAAAAGATATTTTGTGGGggatt 401 17  47, 073, 229  47, 073, 528 YGAGGGGGTTTTAGGGT 402 2 198, 650, 889 198, 651, 188 ACCCTAAACCTACCACCTAAC 403 13 109, 147, 667 109, 147, 966 TCCTCCTACTCCCAAAATCT 404 Antisense Chr Antisense sequence SEQ ID NO 2 AATCCRCTAAAACTCTAAAATAACACCC 405 19 TTGTTGGTATTTTTGTTTTTGGG 406 7 CTCAAATCTCAATTACTCTCAAAATAAA 407 4 TGTTGATAGGTGTAGGTAGGATAG 408 7 CCACCAAATTATTACATAAAATCTACAA 409 7 AAAACRCCCCAAACACCAAATAAAA 410 1 TTTTGGATTGGGTTAGGGTATT 411 15 GATTTTYGGGGTGGGTGGG 412 13 ACAACRATTTCCAAATTCCTACTAAC 413 7 RCTCAAATCTCAATTACTCTCAAA 414 8 TGATTGTAAGYGTAGGTTTGGGT 415 18 CAACAATACACTATACRACTCCTACAA 416 6 ACCCCTTTCRCTCCCTTCCTA 417 2 AGAGATGGGAAGAGAAAGTGG 418 6 TTTTATTTTTYGTGAAAGTAATGATATAGTAGAA 419 8 ATCCTAACCCTAAAATCCCTAAAT 420 6 CCTTCTCAATAAATACATTTACCCC 421 8 AATAACCACCACCRCTCCTCC 422 5 RCCACCTTTCTTAAATAACTCT 423 10 CCCCAAATCAACCCTTTTTC 424 11 RCCTATTTATAATAATAATCAAACCCA 425 19 RATCCCACCTACTAATATTCC 426 2 ACCTCRACACCACCAAAAATAAA 427 22 TGAGGGGAGATTTGAGGGAT 428 11 AATTCCCRAACCCCCTCCC 429 8 GGAAAATTTTTAGTATTTGAGAATGGA 430 2 TCRAAATCCCAAAACCACAACC 431 6 TTTTTGGGATTGTGGTGGAG 432 20 GGGTGGATYGTGGGTTAGTTTT 433 7 AACCACCACTAAAACCCTAAA 434 5 GGTGTGTGTGTGTAATATAATAATTTGT 435 3 ACAAACCAACCACATTTACTTCT 436 12 AAAACCACCTTCCTAACTCC 437 7 ATGGAGGTTTAGGTYGGTGTAAA 438 8 TTATGGGGGYGGGTGATGGA 439 11 ACTTCCCRCCCAACCAACTTC 440 7 ACCCATCTCCCCRAAAACTAAT 441 4 GAAATGAGTTTGGTAGGTGGTT 442 8 GGTGGAGGAGGTGGTGATTA 443 10 YGTTGATTATGGTTGGTTTGTT 444 1 GAGGGGTTTTGAGGGTTGTA 445 15 TGAATTTTATTGTTATGTGGGGTATT 446 1 RAAAATACACCTAAAACAAAAACTAT 447 2 ACTTTTTAACATCTATTTTACTAACCTATT 448 5 CCCTTTCCCRCTCCACTACTC 449 3 GGGTGGTGGTAATTTGGTT 450 7 TGGTTGGGYGGTAAGTATTATGTTG 451 10 AGGGTTAGAGTTTTTGGGTTAG 452 8 CCCTCCCRACCAAAAACTCA 453 12 ACAACAACCRAATAATCCCCATTC 454 3 TTATATTTGTTTTTGGGAGGAGTG 455 11 TGGTTYGGATTGGGGTAGGAT 456 6 CCTCCCRACCCTCCTCCC 457 2 TCCTAATTTCTTACCTCATTACACT 458 11 ACAAAATAAAAACTCCRAAAATAAAATCCC 459 1 GTGGTGTYGGTTTTTAAGGGTT 460 14 ACCCAACCCTACTTAACTCTC 461 7 ACCCRACTTCCTTTATCCCCA 462 10 ACTAAAACCCCTAAACCAACC 463 1 GGTGGGTGGGAAGTAGGAT 464 4 RAAATAAATCTAATAAATAATTTTCCCC 465 7 RACTTCCTTTATCCCCAATCTa 466 2 TCTCTCCTCCCCAACCRTCTAAAA 467 3 ATCAAATTCCCCAAAACCCT 468 7 TTAAAAATCCCCACCAACAAC 469 14 GGTTAGTAGGTTGTTTAGGAGG 470 13 RTAAAACTAAACTCCAACTCCC 471 1 TTGGGAGTYGGGGTGGTTAG 472 14 CAATCCCTATAACCCCCTCC 473 20 YGATTTTAGGTTTAGGGTGAATTTT 474 7 CCACCCACCTCTACCTAAT 475 2 AAAACTAATTCCTACRAATTCCTCCTA 476 5 AACCTCTTTAAAACCTTCCCTAA 477 2 AGAAATATTTTAGTGTGAATTAAATAAGTTG 478 10 tgggggtaggggGAGTT 479 20 GTTGTGGGYGGGTAGGAGGT 480 11 TGGGAGGGATTYGAGTTGGTTG 481 1 CCACCAAACCCRCCAACTACC 482 13 TAGGTTGGGTAAAGGAAGGA 483 6 GGTTTTTATTTTTYGTTTTTATTTGATGAGTT 484 2 TCAATCCAACTCTACTCACCAT 485 11 YGTTTGTTTATAGTGATAATTAGGTTTA 486 8 CCTAACCAAAAACCRAATTTAATTTAACC 487 15 ACCTCCCCATACTTTAATCCT 488 6 AAGTTTGGGAGTAAGGAGGG 489 12 TTTTGGTTTTYGGTTTGGAGGG 490 4 AAAACCRAAACTAAATAACAAAATAAACTTC 491 6 CAAACCAAAACCCAACATCAC 492 7 AACCCTACAATTAAACACAAACAT 493 14 RCAAACACCTACCAAAACCA 494 13 GTAGTATTAGYGAGTTTATTAGGAAGGAG 495 4 GAGGTTGAATGGTAAAGTAGGTT 496 10 AAATAAATTTCCAAAAACCAAACAAAA 497 7 TGAGTATAAGTATGTTGTATGGGG 498 14 YGTTTTAGGTTTAGGAAGTTGAATG 499 1 CTCTCCCCCTCCCTAAAAAT 500 10 CACCTCCTCCCRAAAACCCT 501 X CCTCCTCTTTCCTCCCAA 502 2 TTTTTAATATTTATTTTGTTGATTTGTTTGG 503 10 TTGGGTGATTGGGGGTT 504 12 CAAAAACTACAAAAACATCRCTAAATATTACC 505 1 GTGGGYGGGAATTTTAAGGGG 506 2 GTTGGGGAGGGTTGGTT 507 5 GAGATAAYGGGGTTTTTGGGAAG 508 5 CCAAATAAAAATAATAATAAAATATTCCAAACT 509 22 GGTTTATATTTAAGGTTAGGAAGAAGG 510 7 AGATGAGGGGAGAGGTGG 511 11 GGAAAGGAGGGTTAYGGGTAAAG 512 1 CCTCCTAAACTAAACTAAAACACTAAC 513 20 ACCCTCTCCTCCTCTCC 514 7 YGGGAGAAGTTTTTGGGTT 515 2 TTGTTYGGAAAAATTGTTTGGGTTT 516 18 GGGTGGGATAGGATAGGGT 517 11 GAGTAGAGTGTAGGTTYGAAGGTAT 518 20 CCAAAACAAAAATCTAACTACRAAAATCC 519 6 AACCTTAAAACCCAAACCAAC 520 7 GAAAATYGATGAGAGGTAGGGTTAAG 521 5 AAAAATCCCAAACCAAAAACTAAC 522 18 GTAGTGTATTGTGTGGTTTTTGT 523 6 TTTGGGTAAGTTTTGAGGAAAG 524 6 CTCCAAAAACACCTTAAAAACAC 525 6 GGAAGTTAGGTAATTTTTGAAGTTTTT 526 2 YGGAGGTTTTGAGTTATGGA 527 7 RAATTAAAACCAAAAACCAAAAAT 528 20 TTTGGTTTTGGTTGAYGTTGAGT 529 7 GGTGGTGGTTTGGGGT 530 11 gggGTAGTTTTYGTAGAGTGGAG 531 7 GGTTATTATTGGGGTTTTGGGTAA 532 5 ACAACACAACAATTTATCAACTACT 533 18 GGTTTAGTTATTAGGGTTTAGTGGT 534 14 ctttcctctctctctctctct 535 6 GGATTAGTGGTTTTGTTTGGAAAA 536 14 GGGTAYGTTAGAGTGTGTTTTATTATTAG 537 2 CCTTCCTCCTAAAACCCTAAAAT 538 10 AATCCTCAAAAATTCTATTCTTAAACC 539 4 ATGTTGGGGGTGGAATTTT 540 20 GGTGGTTAGTGTATTGYGGAGTTG 541 7 GTTAGGAGTTAGAAGTTGGTGT 542 8 TTTTAGGTGATTGYGAGGTAATTTGT 543 1 GTTGTAGTTGTTTTAYGGTATTGTTGA 544 6 aaaaccccaataaatttcaaatcc 545 7 AATGGGGATYGTAGGGTGGG 546 10 GGATTTTTAGGGATTAAGTAAAGAAATTAT 547 7 CAAACCCTCCACRCTTCTACAAAAA 548 2 TGGTTTAGTTTGGGYGGAGAGTA 549 2 GGAAGGGGGATAGGGGAT 550 6 AAAACAATAACACAACAAAAACCA 551 8 GGTTTYGTGTGGTTGGGGG 552 14 TGAGGTTTGTAGTGAAGGGT 553 15 TGGTYGTGGATTTGATTTTTGTAGT 554 8 AGAGGATATGTAAATTTTTAGAATGTTG 555 8 GTGYGATAGGGATTTTTATTTTTAGGTTTA 556 12 GGGAAGYGGGGTTGTAGGTA 557 7 GGAGATGGGGGATATTTTAYGTTAGTT 558 19 TTGATGTTTTGTAGGGATGGA 559 5 CCTACCCCCTACTTCCCA 560 13 CAACACTAACRAATCCACCAAAAA 561 1 TGTATTTGAGGTAGAAGTTGTGG 562 4 GTTTAGGTGATTTTGATTTTAGGTTTT 563 3 TCCAAAACTTTCAACACCATAC 564 4 AACCCTAATCCCAAATTCCC 565 6 CTCCACCTCCRTTCCTAAAACTATAATAA 566 12 AGTTTTATTTGTAGTAGTYGAATGGTTTT 567 18 YGGAGGTGGGGAGTAGTT 568 8 CTAATAAAATCTTAACTACTCCAAATCAA 569 2 GTGAGAAGGAGAGGGGAG 570 20 TCCAAATCCCCRAAACCCTC 571 6 GTTTAGYGTAGATGGGGTGGGA 572 7 GGTGATGAGGTTGATGTAGTGT 573 7 AATTATTTTTAAAGTGGGGGTAGTAT 574 5 ggAGGGATAGGAGYGAGAGGG 575 2 ACCCAAAACTCAATCTTACTTCT 576 4 ATAAATCTAATAAATAATTTTCCCCACC 577 20 CCCCAAACCCAAAATAAACTTC 578 1 CACCTACCAAATTTACAAATCCC 579 7 CCCRAAACCCCACACATACTT 580 2 TTTGGGTGGAGGTTATGGA 581 15 AACAACCRAACCCACCAAAAA 582 15 ACACTCRACACACTTAAAACAAAC 583 3 GAAGGAGAGGGGTTGGG 584 5 GGTTTGTAGTTTTGGTTATAGTTGT 585 7 GAAGGTAGAGAATTTGGGTTTTT 586 14 TTGAGTGTGTTAGGYGTTTGTTG 587 2 RCTCCAATAACCCAACCTAA 588 20 CCTATTTCTACCCCCACCC 589 8 CCCTTCTCRATCAAAAACATCCTAC 590 16 AATCAACRACTTAAACATTCAAATTAAACT 591 13 GTTGYGTTTGTTAGTGTAGGAAGTT 592 2 GGAAAATTGGAGGTAGGGG 593 7 GGAGTGATTTATGYGTTATTGTTTTGT 594 22 AAACTACACCCRAAAACCTCTAAAT 595 22 GAGGTTTTTATAGGGTAGGATTAGT 596 22 AACAACAACTAATCCRAACTAATAAAAAC 597 1 AAAACCACCTACTAAAACCRAAAATAA 598 6 GGGTAAAGAAGAGGAGAAGATAAA 599 1 RCCAAACCCAAACAAAACA 600 2 ATTCCCAATAACATATTCATTTACAAAA 601 7 CATAAATCAAACCRCAAAAATCTCCAATA 602 2 CCTCCTCCCRCATAAAAATATCAAAATAA 603 1 ACTACAACTACTCTACRACACTACC 604 5 ggAGGGGTYGTTTTAGGGGG 605 7 ACTAAAACCCRCACCTAACCCA 606 14 GTTTGGGTGATAATAGGGAAAATTA 607 10 CTAACCATAACTAACCTACCCC 608 3 CCTTCAAAACCAAATACAAACTTAT 609 11 aaagaagaagaagaagaagaagaTTAG 610 10 YGTTGATTATGGTTGGTTTGT 611 11 GATAGGGATTTYGGGGATAGGG 612 11 CTACAAATAAAACTTCTTTCCAAATAAAC 613 5 GAGTYGGGTAGGTTTTTGGGTT 614 2 GAAGATGTTAGGGTAGYGAGTTTTG 615 14 CAAAACAATAATTTCTCAACTTTTCC 616 1 AATACAACATAAAAACTCTTTCRCTAACAC 617 11 ACTCCCTCCCTTCTATTTTCA 618 4 AATACAAACAAAACAATCCCTCCAC 619 7 ACTTTTACTATAAAAATTATAACTACAAAACATC 620 19 ATCAAACTATCCCTAACCRAAATTCTA 621 5 TGAAGTAATGAGATGAAAAGTATAAGAG 622 2 RAAAACTTAAACCAATCCAAC 623 15 TGGTTGTGGAGGAGTTGAG 624 5 TTTTGTGTGGGAGTTGGTT 625 14 ACATATTTACTACATTTCCRACCTAAAC 626 10 GGTTTTTAGGAGTTTTGTTTTTAGAT 627 19 GGAGGAGATGTTGTTTTTAGTG 628 6 GTTGTTAAATAAAAAGTYGGGGTGAG 629 20 ACTTTTTATCTCTTACAAACRTCTCCTAAAC 630 7 GGGTAGTAGTGTGTGTAGGG 631 5 ACCTACAACCCTAACTACAACTA 632 7 CAACTCCTCRAAATACCCAATACA 633 14 CAACCCCAAAACCAACAAAT 634 20 TTTTTAGYGGGATAGGGTGTTGG 635 18 GTTAGGAGGGATTYGGGAGGT 636 11 AAACCCTTCCCAACCCT 637 9 ACTCCATCAACRACATCCTAAACA 638 1 yggtgttaggttgtgggt 639 18 GGAGGGGTGGGATAGGA 640 1 TGGTTGTAATAGGYGGTGGGT 641 7 TGGGTATGYGGGTGTTTTAGGA 642 11 CATCCRCTAACCAATAAACTTCCTTAAA 643 7 CTCCCACCCCTAAACAAAC 644 X GTGGGTGTGGGAGGTTT 645 1 rataccaaactataaacaaaccc 646 7 CCCCTATACCTCTATCTCTACC 647 2 GTGTTGTGGGGGTTTTGG 648 2 GGGTGTTAAGATAAGATATGTTTAGT 649 11 ACCAAACCCTAAAACAACAAAT 650 14 GGGTTAAATAGAAGAAATGTTTTTAATG 651 1 RCTAATTCCTAACAACTAAACCAAC 652 17 GTTTTTGGGGAGGGGGAA 653 8 ACCCCTATCCCCAAATCTAC 654 17 TCCCTCRACTTTAACCAACTCA 655 2 GTGGAGGAGAGGGGAAG 656 13 YGTGGGGTTGAGTTTTAGTT 657 Table 3 below provides statistical information of exemplary assays involving all 150 MVPs as defined in Table 1 above, the top 3 ranked MVPs (SEQ ID NOs:1-3), the top 5 ranked MVPs (SEQ ID NOs:1-5) and the top 10 ranked MVPs (SEQ ID NOs:1-10).

TABLE 3 All 150 Top 3 Top 5 Top 10 Sensitivity 0.93 0.61 0.66 0.70 Specificity 0.97 0.71 0.74 0.80 PPV 0.98 0.79 0.82 0.86 NPV 0.89 0.51 0.55 0.61 AUC 0.95 0.66 0.70 0.75

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The invention claimed is:
 1. A method, comprising measuring from a biological sample from an individual the methylation status in each one of a group of Methylation Variable Positions (MVPs) selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein the measuring identifies the presence of methylation in the [CG] of each of SEQ ID NOs 1 to
 3. 2. The method of claim 1, wherein the group of MVPs comprises at least 40 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].
 3. The method of claim 1, wherein the group of MVPs comprises at least 50 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].
 4. The method of claim 1, wherein the group of MVPs comprises at least 100 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].
 5. The method of claim 1, wherein the group of MVPs comprises all of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG].
 6. The method of claim 1, wherein the MVPs determined to be methylated include the MVPs identified in SEQ ID NOS 1 to 3 and denoted by [CG], or include the MVPs identified in SEQ ID NOS 1 to 5 and denoted by [CG], or include the MVPs identified in SEQ ID NOS 1 to 10 and denoted by [CG], or include the MVPs identified in SEQ ID NOS 1 to 40 and denoted by [CG].
 7. The method of claim 1, wherein a treatment for bladder cancer is administered to the individual when at least 25 of the MVPs of the group are methylated.
 8. The method of claim 1, wherein a treatment for bladder cancer is administered to the individual when at least 40 of the MVPs of the group are methylated.
 9. The method of claim 1, wherein a treatment for bladder cancer is administered to the individual when at least 50 of the MVPs of the group are methylated.
 10. The method of claim 1, wherein a treatment for bladder cancer is administered to the individual when all of the MVPs of the group are methylated.
 11. The method of claim 1, wherein a biological sample obtained from the individual is a sample of urine, blood, serum, plasma or cell-free DNA.
 12. The method of claim 1, wherein the measuring step comprises bisulphite treatment of DNA from the sample, amplification of the group of MVPs using methylation specific PCR primers, sequencing, and/or using methylation-discriminatory microarrays.
 13. A method of analyzing urine or a bladder sample from a patient, comprising the steps of: assaying from the urine or bladder sample the methylation status of each one of a group of Methylation Variable Positions (MVPs) selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein the assaying identifies the presence of methylation in the [CG] of each of SEQ ID NOs 1 to
 3. 14. A method of treating a patient for bladder cancer comprising administering bladder cancer therapy to a patient after being determined to have at least 25 Methylation Variable Positions (MVPs) methylated, wherein the MVPs are selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], and after being determined to have methylation in the [CG] of each of SEQ ID NOs 1 to
 3. 15. The method of claim 14, wherein bladder cancer therapy is administered to a patient after being determined to have at least 40 MVPs methylated.
 16. The method of claim 14, wherein bladder cancer therapy is administered to a patient after being determined to have at least 50 MVPs methylated.
 17. The method of claim 14, wherein bladder cancer therapy is administered to a patient after being determined to have all of the MVPs methylated.
 18. The method of claim 14, wherein the cancer is non-muscle invasive bladder cancer (NMIBC) or muscle invasive bladder cancer (MIBC).
 19. A method, comprising measuring the presence of methylation in the Methylation Variable Positions MVPs identified in [CG] of each of SEQ ID NOs 1 to 3 in DNA from a urine sample from an individual, said presence of methylation relative to a non-cancer control, wherein the measuring identifies the presence of methylation in the [CG] of each of SEQ ID NOs 1 to
 3. 20. The method of claim 19, further comprising measuring from the urine sample the methylation status in each one of a group of MVPs selected from a panel comprising the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG], wherein the group comprises at least 25 of the MVPs identified in SEQ ID NOS 1 to 150 and denoted by [CG]. 